Probes, Methods of Making Probes, and Applications using Probes

ABSTRACT

Provided herein are methods and apparatuses for analyzing molecules, particularly polymers, and molecular complexes with extended conformations. In particular, the methods and apparatuses are used to identify sequence information in molecules or molecular ensembles, which is subsequently used to determine structural information about the molecules. Further, provided herein are various methods of forming probes and films for making such probes of nanoscale dimension.

TECHNICAL FIELD

The present invention relates to methods and apparatuses for analyzingmolecules, particularly polymers, and molecular complexes with extendedconformations. In particular, the methods and apparatuses are used toidentify sequence information in molecules or molecular ensembles, whichis subsequently used to determine structural information about themolecules. Further, the present invention relates to forming probes andfilms for making such probes.

BACKGROUND ART

Twenty-first century science and technology endeavors, research anddevelopment innovations that solve problems for man-kind willincreasingly be dominated by the ability to make structures and objectsthat have sizes with length scales approaching those of atoms andmolecules having dimensions of a nano-meter or less. Nano-scale matterand objects exhibit unique behaviors, some of which have yet to beunraveled in addition to the known remarkable optical, thermal,electrical and mechanical properties. These open new vistas for manybeneficial applications making them suitable for many applications. Forexample, sequencing, imaging, nano-lithography, manipulation, nano-scaleself assembly, nanometer scale chemistry, and infinite otherapplications with benefit from nano-scale technology development.

It is envisioned and believed that being involved in the nano-sizefrontier of science, technology and innovation is a sure path toregional and national economic well being, and competitiveness. This isevidenced by the extraordinary investment activities by big and smallcountries, large and small private sector enterprises and nearlyunparalleled entrepreneurial activities.

To advance in the nano-scale frontier science and technology requiresaccess to and mastering the following:

Tools to produce nano-objects

Tools to measure sizes with sub-Angstrom precision

Substrates that have atomic smoothness with minimum contamination

Tools to see (image) nano-objects and manipulate them, grabbing, moving,gluing, etc.

Nano funnels/nozzles/probes for dispensing substances and stimuli

Tools to accurately measure all physical properties, thermal,electrical, optical,

Key parameters become smaller by 10 to 20 orders of magnitude ofquantities accustomed to in the macro-world.

In the last 5 years the collective achievements of the best andbrightest people around the world related to the above tools have grownat astonishing rates, delivering numerous discoveries, innovations,methods, products and tools.

One area that could tremendously benefit from nanotechnology is thedevelopment of high-throughput DNA sequencers in the 1990's have helpedlaunched the genomic revolution of the 21st century. Almost on a monthlybasis, one research group or another is announcing the completesequencing of a biologically important organism. This has allowedresearchers to cross reference species, finding shared and/or similargenes, and allowing the knowledge of molecular biologists in all thevarious fields to come together in a meaningful way.

However, current techniques in DNA sequencing are far too tedious, tyingup the valuable time of researchers. Even the fastest, most advanced DNAsequencers can at most process a few hundred thousand base pairs a day.The Human Genome Project took over 10 years to complete, indicating thatcurrent DNA sequencing technology still has a long way to go before itcan be used as a diagnostic tool. Considering that there are about 3billion DNA base pairs in the mammalian genome, and current sequencingtechnology is capable of sequencing about 2 million DNA base pairs perday, it would still take over 4 years to sequence the human genome.

Known nucleic acid sequencing methods are generally based on chemicalreactions that yield multiple length DNA strands cleaved at specificbases. Alternatively, other known nucleic acid sequencing methods arebased on enzymatic reactions that yield multiple length DNA strandsterminated at specific bases. In either of these methods, the resultingDNA strands of differing length are then separated from each other andidentified in strand length order. The chemical or enzymatic reactions,and the methods for separating and identifying the different lengthstrands, usually involve repetitive procedures. Thus, there remainssignificant limitations on the speed of DNA sequencing usingconventional technology.

Despite these limitations, an incredible collaborative heroic effort wasundertaken for the Human Genome Project. It took many years and billionsof dollars to obtain the sequence to the human genome. It would behighly desirable to provide a method and system that reduces the timeand effort required would represent a highly significant advance inbiotechnology. Indeed, frontier advances are required to increase theefficiency and speed of DNA sequencing if we are to expand the genomedatabases that presently exist to include a genome library includingflora and fauna. Certain flowering plants have 100 times more base pairsthan the human genome, so existing sequencing technology must be leapedfor a new frontier of sequencing systems.

Pores

One particular type of sequencing method relies on passing strands ofDNA through pores. For example, U.S. Pat. Nos. 5,795,782, 6,015,714,6,267,872, 6,362,002 6,428,959 6,465,193 6,617,113, 6,627,067,6,673,615, 6,746,594 6,870,361 describe various sequencing techniquesand apparatus based on pores and flow of DNA fragments through pores. Ingeneral the prior art pores have thickness that cannot directly resolvewith high spatial resolution without some other indirect deconvolutionof the date resulting from changes in ionic conductivities. It furthercannot be used for large DNA fragments. Further, it is very timeconsuming. In general, for an ultra fast DNA sequencing system, thereare many limitations with pore based systems.

Therefore, it would be desirable to provide an improved system andmethod of analyzing extended objects such as linear polymers (includingproteins, DNA and other biopolymers).

OBJECTS AND BRIEF SUMMARY OF THE INVENTION

The present invention teaches new methods, devices and tools thatadvances the nanotechnology art listed above. By departing from methodsof prior art and adding new techniques to improve prior art, theteachings of the present invention result in:

The ability to make free standing nano-thickness atomically smoothfilms, including single or multiple layers from graphene, mica, and fromother layered materials.

These atomically smooth layers can be used as substrates fornano-precison tools

Novel methods to handle the layers, the low cost the production of openand closed nano-probes, funnels, tweezers become possible.

The thickness of the layers are used advantageously to defines thenano-scale dimensions of objects.

The nano-probes in combination with other elements, are used to maketools nano-scopes, to recognize and analyze objects

The novel tools exceed the capability of AFM and STM in the theirability to sequence DNA, RNA more rapidly

Novel nano-lithography tools are produced using the thicknesses of thethin-film layers to define the smallest dimension.

Accordingly, in one aspect of the invention an object is to producesingle mono-atomic layers of graphene or mica and other layeredmaterials conveniently and inexpensively. Another object of this aspectof the invention to separate or exfoliate single mono-atomic layers fromlayered materials such as graphite, mica, dichalgoenides, and attachingthem to substrate through a releasable bond.

In another aspect of the invention, an object is to produce atomicallysmooth layers of metals, insulators, semiconductors, organic andbio-molecular layers.

In another aspect of the invention, an object is to produce andmanipulate fibers, organic and bio polymer, nano-tubes and otherstructures.

In another aspect of the invention, an object is to make alternatingheterogeneous layers.

In another aspect of the invention, various probes are formed having tipactive area dimensions that are a measured based on a film thicknessduring manufacturing.

In another aspect of the invention, various probes sets and arrays areformed using the above mentioned probes.

In another aspect of the invention, method of analyzing extended objectsare provided using the herein described probes, probes sets and probearrays.

For example, using the herein nano-nozzles, a DNA sequencing method ispresented that may sequence the entire Human Genome in a matter ofminutes. Realizing and optimizing this technology opens new vistas forhuman endeavors, and enables practical applications that are nearlylimitless. Culturing bacteria would be a thing of the past. Wheneverfaced with an unknown organism, not only could its exact species bedetermined immediately, but also its entire genotype, including newmutations or signs of genetic engineering. This process is based onutilization of the nanoscale probes, e.g., in the form of electrodes,nozzles, funnels, or other suitable probes. These nanoscale probes arecoupled with detection of ultra small and ultra fast signals. This setsthe course for the development of the ultimate sensor, not only for DNA,and RNA, but also to sequence denatured proteins (amino acid sequence ofpolypeptides).

As discussed above, current DNA sequencing technology is most oftenbased on electrophoresis and polymer chain reaction (PCR). PCR is usedto create varying lengths of the DNA in question, which is thensubjected to electrophoresis to resolve the size differences between theDNA fragments. However, this technique faces several bottlenecks. First,although PCR is useful in amplifying the amount of DNA material, it istime consuming, requires numerous reagents, including the use of anappropriate primer. Second, electrophoresis speed is dependent on theapplied voltage. But the applied voltage cannot be further increasedunless heat dissipation is similarly increased. Also, electrophoresisgel is only capable of resolving a small dynamic range (<500 bp). Thisrequires splitting an organism's genome apart for sequencing and thenre-assembling the pieces.

Instead of relying on electrophoresis to resolve the DNA sequence, theproposed sequencing technology is based on nano-electronics.

The herein system and method relies on probes having resolutioncapabilities less than the dimensions of the objects to be analyzed.Further, systems and methods are provided herein that allow for accuratemeasurement of the portions of the specimens to be analyzed, such asindividual monomers in a polymer chain.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary as well as the following detailed description ofpreferred embodiments of the invention will be better understood whenread in conjunction with the appended drawings. For the purpose ofillustrating the invention, there is shown in the drawings embodimentswhich are presently preferred. It should be understood, however, thatthe invention is not limited to the precise arrangements andinstrumentalities shown. In the drawings, where:

DETAILED DESCRIPTION OF THE FIGURES

Described herein is a novel system and method for analyzing extendedobject specimens. The system includes analytical probes configured anddimensioned such that the edge of the probe has a thickness directionthat is spatially smaller than the desired resolution. Further, incertain embodiments, the analytical probe has a width dimension that ismuch larger than the thickness of the extended object. In otherembodiments, the analytical probe has a path in the width direction thatis much larger than the thickness of the extended object.

The “extended object” to be analyzed using the probes described hereinmay be a complex macromolecule, including complex monomers, polymers,oligomers, dentimers, or other molecules. Examples of such complexmacromolecules include, but are not limited to, proteins, polypeptides,peptide-nucleic acids (PNA), having a polypeptide-like backbone, basedon the monomer 2-aminoethyleneglycin carrying any of the fournucleobases: A, T, G, or C. In certain embodiments, the polymers arehomogeneous in backbone composition and are, e.g., nucleic acids orpolypeptides. A nucleic acid as used herein is a biopolymer comprised ofnucleotides, such as deoxyribose nucleic acid (DNA) or ribose nucleicacid (RNA). In certain embodiments, the extended object is a singlestranded (denatured) DNA molecule with a rigid structure. Other organicor inorganic molecular structures may also be extended objects for thepurpose of the present invention whereby these extended objects may beanalyzed, manipulated, physically altered or chemically altered.Further, double stranded structures may be analyzed according to certainembodiments herein, such as double stranded helical DNA strands.

It will be appreciated by one skilled in the art that the systemdescribed herein for monomer level resolution may be used for othermolecular level detection, e.g., for single small molecules, singlemonomers, oligomers, or other nano-scale structures.

Further, as used herein, the term “probe” refers generally to any deviceused to interact with individual portions of the extended objectincluding, for example, individual nucleotides of a RNA or DNA strand,atomic groups an extended object, atomic and molecular bonds and bondinteractions, groups of atoms or molecules within the extended objects,and other interactive forces such as covalent bonds, hydrogen bonds,ionic bonds, and other know interactions. Probes may be formed ofvarious configurations and materials to be described further herein.

Further, as used herein, the term “detectable interaction” refersgenerally to an interaction between the probe and a portion of theextended object. The portion of the extended object with which adetectable interaction occurs may include individual atoms, molecules,or groups of atoms or molecules, and their bonds. The detectableinteraction may be in the form of electric field, magnetic field,optical variations, vibration forces, gravitational forces, or othermeasurable events.

The probes used herein may be formed of various materials andconfigurations. For example, probes may be in the form of wells, nozzlesor funnels (herein after “hollow probes”) having a tip for dispensing orholding materials (including solids, liquids, gases and transitionphases) to facilitate analysis of the specimen. Alternatively, the wellsor nozzles may be provided in a system and configuration for suction orapplication of fluid pressure. The nozzles configured for dispensingmaterials may include conductive inner walls, or a conductive elementdisposed within a material holding region, in order to facilitatemeasurement and other voltage applications across the probe. In otherexamples, the dispensing materials are within a conductive medium tofacilitate measurement and other voltage applications across the probe.

Referring now to FIG. 2, a continuous edge probe 202 is depicted, forexample, in the form of a continuous knife edge. Probe 202 isparticularly well suited for analyzing extended object specimens such asbiopolymers. Probe 202 is characterized by a tip 204 thickness t, a tip204 width w, and a height (not identified in the Figure). Importantly,the tip thickness t is dimensioned to obtain the desired resolution ofthe system. For example, when information regarding individual monomersof a DNA strand is desired, the thickness t should be less than thenucleotide spacing on the strand (about 0.5 nm). Still further, probe202 has a width dimension w that is preferably much greater than the tipdimension and also much greater than the width of the specimen. Incertain embodiments, this width dimension that is much greater than thetip dimension minimizes or eliminates landing error associated withtypical probe analysis systems as the probe passes over the specimen.The ratio of w to t may be, for example, on the order of about 5:1.10:1, 10 s to 1, 100 to 1, 100 s to 1, 1000 to 1, 10,000 to 1, orgreater depending on the desired application.

These continuous edge probes may be hollow, solid or partly solid andpartly hollow.

As shown, in certain preferred embodiments, the probe has a shape thatprovides a larger end 206 opposite the tip 204. This can, for example,reduce electrical resistance of the probe when end 206 serves as acontact region. Further, the larger end 206 serves to facilitateintroduction and dispensation of materials from the probe when the probeis in the form of a nozzle filled with suitable material, as describedfurther herein.

Referring now to FIGS. 2B and 2C, discontinuous probes 222, 242 areprovided. Probes 222, 242 have an elongated width structure withdesirably sized tip with, e.g., cutouts or discontinues edge portions.The generalized probes, 222, 242 made according to the present inventioncan be made in a configurations that several probe sections, 230, 250,which can be accessed independently or together as shown in FIGS. 2B and2C. In certain embodiments, the probe sections 230, 250 serve identicalfunctionality, for example, for redundancy, or to examine pluralspecimens in parallel. In further embodiments the probe sections 230,250 serve different functionalities. For example, some applications mayrequire that sub-probe 230 a be used for analyzing or sequencing thespecimen, adjacent section, 230 b used for dispensing substances orstimuli, and section 230 c used for imaging or reading alignment marks.In another example, probe section 250 a is in the form of an edge withan elongated width as shown, while probe section 250 b may be point likeprobe, as represented in FIG. 2C with dotted lines. The probe sectionsmay be functionalized differently to recognize parts of a specimen undertest with high degrees of specificity. These discontinuous edge probesmay be hollow, solid or partly solid and partly hollow.

Referring now to FIG. 3, a probe 302 is depicted. Probe 302 isparticularly well suited for analyzing extended object specimens such asbiopolymers. Probe 302 is characterized by a tip thickness t, a tipwidth w, and a height (not identified in the Figure). Further, probe 302is positioned within a suitable sub-system 308 to impart motion to theprobe generally in the direction of the width w along a path pw. Similarto probe 202, the tip thickness t is dimensioned to obtain the desiredresolution of the system. The width dimension w of probe 302 is notcritical. However, the path width pw is preferably much greater than thewidth of the specimen. This ensure that as the probe passes over thespecimen, landing error associated with typical probe analysis systemsis eliminated.

The probes described herein may take on various shapes andfunctionalities. In certain embodiments, the probes herein have acontinuous edge that is closed. In certain embodiments, the probesherein have a discontinuous edge that is closed. In certain embodiments,the probes herein have a continuous edge that is open. In certainembodiments, the probes herein have a discontinuous edge that is open.In certain embodiments, the probes herein have a continuous edge thathas some portions along the width w of the probe that are closed andsome portions along the width w of the probe that are open. In certainembodiments, the probes herein have a discontinuous edge that has someportions along the width w of the probe that are closed and someportions along the width w of the probe that are open.

Note that the probes herein may have a constant cross section along thewidth w of the probe, or in certain embodiments, it may be desirable toprovide a cross section along the width w of the probe that is differenttherealong, for example, with a broader or narrower central portion.

Further, the probes herein may have a constant tip opening or tip activearea dimension along the width w of the probe. Alternatively, in certainembodiments, it may be desirable to provide a tip opening or tip activearea dimension along the width w of the probe that is differenttherealong, for example, with a smaller and larger sections of tipopening or tip active area dimension for different applications.

Additionally, the probes may be formed of a generally inactive bodyportion, and an active area that forms the tip opening, such as aconductor in the case of closed tip probes, or a tip opening.Alternatively, the body portion may incorporate some otherfunctionality, such as thermal and electrical shielding, precisemetrology spacing, or other elements such as micro- or nano-fluidic ormicro- or nano-electromechanical devices. Further embodiments will bedescribed herein.

The probes described herein may be formed many different shapes thatwill provide the desired tip characteristics and dimensions. FIGS. 3A-3Lshow various shapes of certain embodiments of probes herein, generallyhaving a closed tip configuration. However, it should be understood thatthese shapes may also be suitable of any tip configuration and may beincorporated in any continuous edge or discontinuous edge probedescribed herein.

FIG. 3A shows a prismatic shaped probe having a cross section in theform of an elongated tip integral with a triangular region and anelongated rectangular portion at an end opposite the probe tip.

FIG. 3B shows a prismatic shaped probe having a cross section in theform of a right triangle, e.g., with the tip flattened.

FIG. 3C shows a prismatic shaped probe having a cross section in theform of a trapezoid.

FIG. 3D shows a prismatic shaped probe having a cross section in theform of a rectangle.

FIG. 3E shows a prismatic shaped probe having a cross section in theform of a triangle, with the tip at the adjoining end of the long sidesof the triangle forming the tip for probing or other applications asdescribed herein.

FIG. 3F shows a prismatic shaped probe having a cross section in theform of a rectangle with a triangle at the probe tip end, with the tipat the adjoining end of the long sides of the triangle forming the tipfor probing or other applications as described herein.

FIG. 3G shows a prismatic shaped probe having a cross section in theform of an irregular polygon, e.g., symmetrical about the height axis,with a flat end and with a tip at the adjoining end of sides of thepolygon with an acute angle as shown.

FIG. 3H shows a probe having a cross section generally in the form of aninverted tear drop, with a tip t at the point of the tear drop shape.

FIG. 3I shows a probe having a cross section generally in an elongatedirregular form, with a tip t at an elongated end thereof.

FIG. 3J shows a probe having a cross section generally in the form of anellipse, with a tip t at a tangential point elliptical shape at anelongated end thereof.

FIG. 3K shows a probe having a cross section generally in the form of anozzle, such as a “flattened” end of an elliptical or circular crosssectioned tube, with a tip t at the “flattened” end thereof.

FIG. 3L shows a probe having a cross section generally in the form of aV-shape, with a tip t at the point of the V-shape.

Referring now to FIGS. 4A-4E, probes are shown in various configurationshaving tip openings t_(o), suitable for dispensing and/or holdingmaterials according to the various embodiments herein.

FIG. 4A shows a probe having a cross section in the form of an elongatedhollow tip integral open to a triangular well region and an elongatedrectangular well portion at an end opposite the probe tip with anopening t_(o), having a channel therein for holding and facilitatingdispensing of materials.

FIG. 4B shows a probe having an asymmetrical cross section in the form arectangle and a truncated triangle forming a probe tip with an openingt_(o), having a channel therein for holding and facilitating dispensingof materials.

FIG. 4C shows a probe having a symmetrical cross section in the form atruncated triangles forming a probe tip with an opening t_(o), having achannel therein for holding and facilitating dispensing of materials.

FIG. 4D shows a probe having a symmetrical cross section in the form aangled members forming a probe tip with an opening t_(o), having afunneling channel therein for holding and facilitating dispensing ofmaterials.

FIG. 4E shows a probe having a symmetrical cross section forming a probetip with an opening t_(o), having a shaped well and a channel thereinfor holding and facilitating dispensing of materials.

Referring now to generally to FIGS. 5A-6B, probes having tips, forexample, conductive tips, with tip active area dimensions of t, areshown, whereby tips 510, 610, extend beyond the bodies 520, 620, of thestructures. As shown, in FIGS. 5A-5B, a symmetrical probe is provided,and in FIGS. 6A-6B, a symmetrical probe is provided. Generally, thedimensions a FIG. 5A and the dimensions a and b in FIG. 6A are greaterthan the tip dimension t, preferably multiples of the tip dimension t.These embodiments advantageously provide for tips that extendsufficiently far away, for example, to minimize interaction between theprobe body, for example, with the specimen or a substrate depending onthe application of the probe. This avoids negative effects of substratematerial such as accumulation of electrostatic charge and otherinterfering effects. Referring to FIG. 7, an example of an array ofprobes according to the embodiment of FIG. 6A-6B is shown.

Referring now to FIGS. 8A-8C, views of an open tip probe according tocertain embodiments of the present invention is shown, showing anirregular inner channel surface. FIG. 8B shows an array of such probes.FIG. 8C shows a probe generally as in FIG. 8A, wherein only a portion ofthe inside surface has electrodes 842 therethrough, which may beadvantageously in certain applications.

Referring now to FIGS. 9A-9B, views of an open tip probe according tocertain embodiments of the present invention is shown, showing anirregular inner channel surface with differing sub-sections therein. Forexample, referring to FIG. 9A, a probe is shown having sub-sections thatare divided generally along the height dimension of the channel,including sub-sections 912, 914, 916, 918 and 920. For example,sub-sections 912, 914, and 920 may be formed of insulating materials,sub-section 916 formed of conductive materials, and sub-section 918formed of semiconductor materials. In a further example, and referringto FIG. 9B, a probe is shown having sub-sections that are dividedgenerally along the height dimension of the channel, includingsub-sections 932, 934, 936, 938 and 930. For example, sub-sections 932and 936 may be formed of conductive materials, sub-sections 938 and 940formed of insulating materials, and sub-section 934 formed as an openchannel perpendicular to the channel of the probe tip, for example, forproviding micro-fluidic operations or other suitable functionality.

In general, variable opening probes may be provided. In certainpreferred embodiments, the opening tip dimension is controllable withsub-angstrom precision.

Referring now to FIG. 10A a sectional view of a variable tip probe 1010according to certain embodiments of the present invention is shown,showing an irregular inner channel surface having a fixed section 1014and a complementary movable section 1016. The movable section 1016preferably are actuated with angstrom or sub-angstrom precision todefine the probe opening 1012. FIGS. 10B1 and 10B2 shows views lookinginto the probe opening according to one embodiment, and FIGS. 10B1 and10B2 shows views looking into the probe opening according to anotherembodiment.

Referring now to FIG. 11, another embodiment of a variable gap probe1110 is shown. An actuator 1124 imparts motion to section 1116 of theprobe, thereby changing the opening dimension of the tip opening 1112.

FIGS. 12A and 12B show an enlarged isometric view and side view,respectively, of a probe set 1230 including probes 1242, 1244, 1246, and1248, and a specimen extended object 1250 upon a platform 1228. Incertain preferred embodiments, polymer strand 1250 is a biopolymer suchas a nucleic acid (e.g., DNA). FIG. 12C shows an enlarged sectional viewthrough any one of probes 1242, 1244, 1246, or 1248. FIG. 12D shows atop view of the base platform 1228, showing an exemplary channel 1252.As shown in FIGS. 12C and 12D, in certain embodiments, a measuringvoltage is applied across each probe 1242, 1244, 1246, 1248, andplatform 1228, denoted by reference numerals 1254 a and 1254 b,respectively. As the polymer strand 1250 passes under an activated probe(e.g., a probe with a measuring voltage applied thereto), detectableinteractions occur as described in further detail herein.

FIGS. 13A-13D show a probe set 1330 formed according to embodiments ofthe present invention. The probe set includes, e.g., a 1×4 array(although it is understood that this may be scaled to any size n×mnozzles) of probes 1342, 1344, 1346, 1348.

In certain embodiments, these probes 1342, 1344, 1346, 1348 are in theform of nozzles, e.g., having tips 1354 associated with wells 1356, asshown in FIGS. 13B and 13C. Generally, the wells having widths in the ydirection greater than the widths of the nozzle tips. FIG. 13D shows asectional view of the nozzle array.

The probe set 1330 may be embedded in a body 1358. The material for theprobes or nozzles, and the body, may be the same or different materials,and may include materials including, but not limited to, plastic (e.g.,polycarbonate), metal, semiconductor, insulator, monocrystalline,amorphous, noncrystalline, biological (e.g., nucleic acids orpolypeptides based materials or films) or a combination comprising atleast one of the foregoing types of materials. For example, specifictypes of materials include silicon (e.g., monocrystalline,polycrystalline, noncrystalline, polysilicon, and derivatives such asSi3N4, SiC, SiO2), GaAs, InP, CdSe, CdTe, SiGe, GaAsP, GaN, SiC, GaAlAs,InAs, AlGaSb, InGaAs, ZnS, AlN, TiN, other group IIIA-VA materials,group IIB materials, group VIA materials, sapphire, quartz (crystal orglass), diamond, silica and/or silicate based material, or anycombination comprising at least one of the foregoing materials. Ofcourse, processing of other types of materials may benefit from theprocess described herein to provide probes and bodies of desiredcomposition.

Referring now to FIGS. 14A and 14B, all probes and probe sets describedherein may be configured with respect to the specimen at various angles.For example, referring to FIG. 14A, a probe set 1430 may be orientedgenerally perpendicular (in the length direction) to a specimen 1450.Further, referring to FIG. 14B, a probe set 1430 may be oriented (in thelength direction) generally at an angle θ with respect to a specimen1450.

Referring to FIG. 14C, a system 1460 is presented whereby theorientation of plural probe sets 1430 relative a specimen 1450 varies.Because the objects of the specimen 1450 (e.g., bases within a DNAstrand) may have different orientations, it may be desirable to sequencewith a plurality of probe sets 1430. The plurality of probe sets 1430may have different angles θ₁, θ₂, θ₃, θ₄, θ₅, . . . θ_(n) (e.g., 20° to160° in suitable increments, arranged sequentially, randomly or inanother desirable arrangement. During measurement as described furtherherein, a controller may determine which orientation of the probe setyields the best signal for a particular base at its inherentorientation. This allows one to measure the data from the probe sets ofthe array, and determine the optimum signal for certain bases or groupsof bases.

In another embodiment, and referring to FIGS. 14D-14F, the angles oforientation in the height direction may also be varied. For example,referring to FIG. 14D, probe set 1430 may be oriented in the heightdirection generally perpendicular (90° with respect to the specimen1450. Further, as shown in FIG. 14E, probe set 1430 may be oriented inthe height direction generally at an angle ω with respect to thespecimen 1450. Referring to FIG. 14F, a system 1470 is presented wherebythe orientation in a height direction of plural probe sets 1430 relativea specimen 1450 varies. Because the objects of the specimen 1450 (e.g.,bases within a DNA strand) may have different orientations, it may bedesirable to sequence with a plurality of probe sets 1430. The pluralityof probe sets 1430 may have different angles ω₁, ω₂, ω₃ . . . ω_(n)(e.g., 20° to 160° in suitable increments, arranged sequentially,randomly or in another desirable arrangement.

In another embodiment, and referring now to FIGS. 15A-15B, the probesaccording to the present invention may be configured about more than oneportion of the specimen to be analyzed, for example, in the form of anextended opening channel which interrogates from more than one side ofthe specimen.

Presently, it is known to coax DNA fragments through a pore for thepurpose of measuring a change in ionic conductivity. Challenges areposed in the consistency of motion through the holes, the resolution,and other interference. The pore is often part of a system of ionicfluids, whereby ionic conductivity change is measured across regions ofionic fluids separated by a membrane and/or layer having one or morepores. For example, as described in the background of the invention,U.S. Pat. Nos. 6,870,361, 5,795,782, 6,267,872, 6,362,002, 6,627,067describe such pores.

However, according to the extended opening channel system 1500 of thepresent invention, a specimen 1550 is passed through an extended openingchannel 1501. Each extended channel opening includes several probesformed according to any one or more of the various embodiments herein.The probes may be configured on one side of the opening, or multiplesides of the opening. In certain embodiments, using an extended openingchannel which interrogates from more than one side of the specimen,accuracy may be enhanced, and signal is increased.

As discussed below with respect to FIGS. 16A-16C, these extended openingchannels may be configured in arrays in a 2 dimensional or 3 dimensionconfiguration, which presently known pore based sequencing systemscannot achieve.

Referring now to FIG. 16A, a serial probe array 1677 is shown. The probearray includes Q serial probe sets 1630. In general, extended objects tobe analyzed may be passed through the Q serial probe sets 1630. The Qserial probe sets may be homogeneous or heterogeneous.

For example, using homogeneous probe sets 1630, each probe set mayinclude various individual probes optimized for adenine, cytosine,guanine, and thymine.

Further, referring to FIG. 16C, an array 1680 of probe sets may compriseheterogeneous probes. For example, one probe set may be optimized foradenine (A), a second optimized for cytosine (C), a third for guanine(G) and a fourth for thymine (T).

These serial arrays would not be possible using conventional knowntechniques, for example, based on pores as described in the backgroundof the invention. Importantly, redundancy is readily achievable in aserial configuration of the present invention, whether the system isformed of serial heterogeneous probe sets, serial homogeneous probesets, or combinations thereof.

Referring now to FIG. 16B, a parallel and serial probe array 1678 isshown. The probe array includes M×N channels of Q serial probe sets1630. This probe array 1678 may be very useful for high speed parallelprocessing of extended objects to be analyzed. The probe sets 1630within the array 1678 may be homogeneous or heterogeneous. The extendedobjects may be the same or different. In general, extended objects to beanalyzed may be passed through the Q serial probe sets. An M×N array ofextended objects, which may be the same or different, are passed throughthe M×N arrays of Q serial probe sets 1630.

The above described probes may be used in various configurations.Certain probes may be in the form of open tip probes. The various opentip probes described herein may be used for dispending materials, forexample, as a nano-nozzle or nano-funnel. Further, various open tipprobes described herein may be used to expose a specimen or a workpieceto photonic energy or stimuli, serve as a as a nano-nozzle ornano-funnel for ion or particle beam operations, or the like.

Further, various open tip probes described herein may be used to exposematerials to a specimen or a workpiece, whereby a) forces are appliedwithin the body of the probe, within the well of the probe, or byanother element within the probe to keep the material from dispensing;b) operate at suitable temperature the reduces the likelihood of orprevents the material from dispensing; or c) operate at suitablepressure the reduces the likelihood of or prevents the material fromdispensing).

Certain probes may be in the form of nano-electrodes for measuringdetectable interactions. Certain probes may be in the form of materialsthat result in detectable interactions such as a system of correlatingbiological materials that create hybridization events with the extendedobject to be analyzed.

In certain embodiments, and referring now to FIGS. 17A and 17B, thebasic principle is described, wherein a DNA chain (or other protein orextended object to be analyzed) 1750 upon a base 1728 is passedunderneath four probes in the open tip probes 1742, 1744, 1746 and 1748(or arrays of nozzles, e.g., as shown in FIG. 17B). The four funnels ornozzles 1742, 1744, 1746 and 1748 are filled with adenine, cytosine,guanine, and thymine molecules respectively. Due to the complementarystructures of adenine and thymine, and of guanine and cytosine, ahybridization event between nucleotides on the DNA chain and thenucleotides in the nozzle will occur when the correct pairs come intocontact. This hybridization results in a lower energy state and chargetransfer, which can be detected via an ammeter. This is because theconductivity between the nozzles and the electrode ground plate will beaffected, thereby altering the current between the nozzle and the groundplate. FIG. 17B shows an exemplary array setup, e.g., that may averageout noise and increase SNR. These features will help in assuring anexcellent SNR.

Note that the above described probes may also be formed with one or moreconductors therein for increase signal detection capabilities. Forexample, the conductor may be layered within or upon an inner wall ofthe probe or nozzle well and tip/

Referring to FIG. 18A, an embodiment of a system 1800 having probesformed of solid state nucleotide materials is shown. A probe set 1830 isdepicted wherein each probe 1842, 1844, 1846, 1848 is formed of a solidstate nucleotide, e.g., adenine, cytosine, guanine, and thyminemolecules respectively. A solid state nucleotide may be manufactured onthin films, and formed as probes using the various manufacturing methodsdescribed herein or other thin film manufacturing techniques.Preferably, these SSN have a single molecule thickness at the probe tip,so that a desirable monomer scale resolution is maintained. These filmsmay be formed in the nozzle wells, e.g., by layering during themanufacturing process prior to slicing. In preferred embodiments of aDNA sequencing system herein, the nozzles are formed with a tipdimension of less than about 0.5 nanometers to resolve correspondingmonomers.

It is known that DNA strands may be condensed on substrates. In theherein probes, single species nucleotide strands may be condensed in theform of lines or films. Referring to FIG. 18B, these may be formed on asubstrate (M), such as a conductive substrate, Referring to FIG. 18C,condensed single species nucleotide strands may be sandwiched betweensubstrates (M).

The films resulting from FIG. 18B or 18C may be used directly as theprobes. Alternatively, these films may be slices and attached tometallic “knife blades”. In a further alternatively, they may be folded,whereby exposed condensed single species nucleotides serve as the probe.

Referring now to FIG. 19, a system 1900 is shown using a metalconductors as probes 1931. The probe may be formed of a suitableconductor material. Further, probes in the form of nozzles may be filledor layered with metal conductor material. The metal may be platinum,gold, or other suitable metal or non metal conductor. In preferredembodiments of a DNA sequencing system herein, the conductor probesformed to a tip dimension of less than about 0.5 nanometers to resolvecorresponding monomers.

In one method of using a probe 1931, stimuli (e.g., a voltage) isapplied across the subject nucleotide within the subject strand, and acharacteristic I vs. V curve may be obtained. For example, FIG. 20 showsan exemplary representation of characteristic curves for variousmonomers adenine, cytosine, guanine, and thymine (A, C, G and T).

In certain embodiments, a single probe 1931 may be used as described inFIG. 19. In other embodiments, a probe set may be used, whereby biaswaveforms across different electrodes may be varied to adjustsensitivity for expected specimen portions or monomers. For example, afour-probe probe set may be used for identifying A,C,T,G components ofbiopolymers such as DNA strands. Further, identical waveforms may beapplied whereby multiple probes are used for redundancy. These may begated or un-gated, depending on the application.

Referring now to FIG. 21, a functionalized group 2150 (FG) is mountedonto probe 2110. The 2150 may include known nucleotide strands,oligomers, peptides, single molecules, or other known species. The 2150is selected to have a known specific sensing capability, for example,electrostatic, magneto-static, chemical, and other interactions with aspecimen under analysis.

Referring now to FIG. 22, a functionalized group 2250 may be attached tocylinders of micrometer diameters which may then be attached to a largerstructures. The cylinders may be coated glass, metal, or organic orinorganic.

Referring now to FIG. 23, plural functionalized groups 2352, 2354, 2356are is mounted onto a probe 2310. In this embodiment, steppingoperations, either of the probe or the specimen, is in two directions.By stepping in a direction substantially normal to the width w of theprobe 2310 and in a direction substantially parallel to the width w ofthe probe 2310, analysis may be simplified. For example, functionalizedgroup 2352 interacts with the specimen, the observation is recorded,then the probe is stepped so that functionalized group 2354 interactswith the specimen, the observation is recorded, and then the probe isstepped so that functionalized group 2356 interacts with the specimen,the observation is recorded. Then, the entire probe may be stepped in adirection substantially normal to the width w of the probe 2310 tocontinue analyzing the specimen.

Referring now to FIG. 24A, an embodiment of a system 2400 having probesformed of conductor with a known material strand attached to the edge ofthe probe, particularly the “knife edge” probe, e.g., described abovewith respect to FIGS. 2 and 3. For example, a probe set 2430 is depictedwherein each probe 2442, 2444, 2446, 2448 has a known nucleotide strand,e.g., adenine strand, cytosine strand, guanine strand, and thyminestrand respectively.

In a preferred embodiment, a single strand/single species nucleotidestrand is provided. It is stretched and attached to the tip of aconductor probe.

The known nucleotide strand may be attached to the tip if the conductorprobe by various nano- or micro-manipulation means.

In one embodiment, magnetically attractive molecules, referred to as“magnetic beads”, may be attached at opposing ends of the known strandto facilitate manipulation. A nano-manipulator magnet system may be usedto stretch the strands for attachment to the probe set. For example,this is shown with respect to FIG. 24B. Further, this configurationensure that as the probe passes over the specimen, landing errorassociated with typical probe analysis systems is eliminated.

With a single-strand, single-species chain attached at the probe tip,when the tip encounters a specimen portion or monomer that is capable offorming a hybrid pair with the probe species, bond energies associatedwith the hybridization event enhances the resonance activity beingmeasuring.

Referring to FIG. 25, an embodiment of a system 2500 having probesformed as open wells or funnels is shown. A probe set 2530 is depictedwherein each probe 2542, 2544, 2546, 2548 is formed as an open well orfunnel. This open well or funnel may be used as a path for various probeactivities, for example, generated by sources 2582, 2584, 2586, 2588.

Particle beam emitters can be made directly into nano probes orindirectly through the funnel described herein. They include ion beamand electron beam emitters.

Photon beam emitters such as x-ray emitters, ultraviolet emitters, IRemitters, visible emitters, and terahertz emitters can be formed withthe herein probes or trough funnels as described herein. In the eventthat the excitation photon beams have wavelengths large than the probediameter, the use of evanescent fields that extend only to the widthscale of the beam (probe) will be utilized.

In another embodiment, an electron beam emitter is focused and shaped toprovide a nano-scale resolution beam. They can be tuned in energy. Thistunability can give one selectivity in directly interacting with thespecimen to be analyzed. Electron beams may be used as the probe for thesystems of the present invention.

It is known in the electron optics art that atomic scale resolution maybe achieved with SEM, TEM, and STEM since the beams themselves can bemade nano-scale as the probing beams. In preferred embodiments of a DNAsequencing system herein, the electron beams are focused to a sectionaldimension of less than about 0.5 nanometers to resolve correspondingmonomers. The electron beam may be a line beam (analogous the probe ofFIG. 2), or electron beam scanning may be employed (analogous the probeof FIG. 3, although it is to be understood that the funnel need not bemoved, only the beam).

Referring to FIG. 25, the electron beam may be inserted through thefunnel. This minimized the need for nano-scale resolution electronoptics required for direct electron beam formation at the atomic scale.

It should be appreciated that the funnel walls for x-ray, electron beamsand ion beams will be constructed appropriately to be able to propagatefrom the funnel opening to the funnel end to achieve nano-scaleresolution. In the case of electron beams, electric fields appropriatelyplaced may cause these beams to bend toward the funnel tip. Alternately,secondary electron emission may be created from inner funnel wallsurfaces which lead to the creation of a beam that exits the funnel tip.

In another embodiment, a focused ion beam emitter with nano-scaleresolution known in the art may be used as the probe to interact withthe specimen. They can be tuned in energy. This tunability can give oneselectivity in directly interacting with the specimen to be analyzed.Further, the ion beams may be based on H+, He+, Ge+, Ga+, or othersuitable ions of substances that may be formed into beams that havespecific selective interaction with the specimen to be resolved.

Referring to FIG. 25, the ion beam may be inserted through the funnel.This minimized the need for nano-scale resolution electron opticsrequired for direct electron beam formation at the atomic scale.

It should be appreciated that the funnel walls for x-ray, electron beamsand ion beams will be constructed appropriately to be able to propagatefrom the funnel opening to the funnel end to achieve nano-scaleresolution. In the case of electron beams, electric fields appropriatelyplaced may cause these beams to bend toward the funnel tip. Alternately,secondary electron emission may be created from inner funnel wallsurfaces which lead to the creation of a beam that exits the funnel tip.

X-ray beams, such as an x-ray laser beam, may be used as the probe forthe systems of the present invention. In preferred embodiments of a DNAsequencing system herein, the x-ray beams are focused to a sectionaldimension of less than about 0.5 nanometers to resolve correspondingmonomers. For example, the electron beam system described above may beused to generate nano-scale x-ray beams in a manner known in the art.

Further, referring to FIG. 25, an x-ray beam (directly or indirectly)may be inserted through the funnel. This minimized the need fornano-scale resolution x-ray and electron optics required for directelectron beam formation at the atomic scale.

It should be appreciated that the funnel walls for x-ray, electron beamsand ion beams will be constructed appropriately to be able to propagatefrom the funnel opening to the funnel end to achieve nano-scaleresolution. In the case of x-ray, the inner surfaces of the funnel maybe made of multi-surface to achieve interference reflection, or may beof single crystal using Bragg reflection properties, or may be grazingincidence angle rejection until the rays reach the funnel end.

To avoid stray x-rays that may interfere with excitation and/ormeasurement and increase noise, the inner and outers surfaces of thefunnel as appropriate may be coated with x-ray absorbers.

Scanning tunneling microscopy(STM) or atomic force microscopy(AFM) probetips may be arranged into arrays and utilized according to the teachingsof the present invention.

The above described probes may be used in various configurations.Certain probes may be in the form of wells with dispending tips. Certainprobes may be in the form of nano-nozzles. Certain probes may be in theform of nano-funnels. Certain probes may be in the form of electrodesfor lithography.

As described herein, for example, with respect to FIGS. 10 and 11,probes may be provided herein with variable dimensioned or actuate-abletip openings. This type of variable gap probe may be very useful formany applications, including but not limited to controlled dispensationof materials, controlled vacuum or fluid pressure, manipulation ofnanometer sized structures, and other applications.

Various configurations of the open tip probes herein may be useful forvacuum or fluid pressure. For example, certain embodiments of the opentip probes described herein may be used to impart vacuum or fluidpressure. In another embodiment, and referring now to FIG. 26, a probe2610 is provided having a plurality of openings 2612 along the length ofthe extended width probe tip, with other regions 2614 plugged withsuitable plug material. The vacuum or fluid source may further bedivided, or alternatively, the plural openings 2612 may share a commonvacuum or fluid source.

Herein disclosed are probes, nano-probes and methods of manufacturingprobes and nano-probes. With the disclosed methods, it is possible tocreate probes with tip active area dimension, such as opening dimensionsin the cases where the probe has an open tip, on the order of about 0.1nanometers to about 10 nanometers, 10 nanometers to about 100nanometers, or 100 nanometers to 1000 nanometers. Further, it ispossible to make such probes in arrays with exact spacing therebetween,and with additional supporting functionality such as stimuli providingstructures, metrology structures, micro- and nano-fluidic structures ordevices, micro- and nano-electromechanical structures, or othersupporting features. Such features enable molecular level dispersion,precise material deposition, molecular level detection, and othernano-scale processes.

Furthermore, the herein described analytical systems includingsequencing of extended objects such as DNA or RNA strands or fragmentsis enabled by creating a probe having tip dimensions on the order ofabout 5 Angstroms, for example, utilizing the herein referenced anddescribed probe and nozzle manufacturing methods. There are variousmethods of making the probes, probe sets and probe arrays describedherein. Co-pending U.S. Non-provisional application Ser. No. 10/775,999filed on Feb. 10, 2004 (and corresponding PCT ApplicationPCT/US04/03770) entitled “Micro-Nozzle, Nano Nozzle and ManufacturingMethods Therefor”, incorporated herein by reference, describe varioustechniques for manufacturing probes in the form of nozzles or funnelsare described. These techniques may be modified to provide other probeconfigurations and probe types described herein.

Further, in certain embodiments, it may be desirable to conduct variousfabrication, handling and assembly steps in clean room environments. Inother embodiments, it may be desirable to conduct various fabrication,handling and assembly steps in a negative pressure environment and/or inultra-pure inert gas environments.

In general, in certain embodiments of the herein described methods ofmaking the films, the probe tip active area has relevant tip dimensions(e.g., tip width t as shown in the above FIG. 2A) that is a function ofa very thin film that is layered, deposited, or otherwise formed eitheron a portion of a probe body or on intermediate structures betweenplural probes.

Prior art teaches how sub-micron objects and features can be produced bymeans of conventional optical, UV, e-beam, X-ray and lithography. Thesetools are being extended to produce sizes below 30 nanometers. As theyare stretched to produce even smaller sizes, their limitations becomemore and more apparent, in terms of cost, foot-print, etc. Indeed, athigh electron and ion beam accelerating voltages >100KV features smallerthat 10 nm have been demonstrated. The preparation steps and the cost ofthe equipment and ancillary components make these prior art methodscumbersome and slow.

The present invention, shows ways to produce similar or better resultsfaster, and more convenient by departing from using lithography basedphoton, ion and e-beams to produce the smallest features. Instead,ultra-thin films are used for this purpose.

There are many known methods of producing films with atomic precision.These include, deposition by sputtering, electron beam, ion beam,molecular beam epitaxy, CVD, MOCVD, plasma, laser deposition, pyrroliticdeposition, electrochemical, thermal evaporation, sputtering,electro-deposition, molecular beam epitaxy, adsorption from solution,Langmuir-Blodgett (LB) technique, self-assembly and many other relatedmethods collectively referred to as Thin Film Deposition Methods.Accurate metrology enables the production and control of thicknesseswith Angstrom precision. Producing free standing films by peeling ispossible as taught in copending U.S. patent application Ser. No.09/950,909 filed on Sep. 12, 2001 and U.S. patent application Ser. No.10/970,814 filed on Oct. 21, 2004 and manipulation taught in applicant'sco-pending U.S. Non-provisional application Ser. Nos. 10/717,220 filedon Nov. 19, 2003 entitled “Method of Fabricating Multi Layer Mems andMicrofluidic Devices” and other related applications. The films producedby the conventional deposition methods need atomically flat substrates.

The advent of scanning tunneling microscopy (STM), atomic forcemicroscopy, AFM, scanning probe microscopy, SPM, and related tools haveenabled the imaging of surfaces and structures with atomic resolution.This opened new vistas to advance our understanding of many physical andchemical phenomena that are being exploited in numerous practicalapplications in the fields of medicine, nanotechnology,nano-electronics, genomics, proteomics, nano-electrochemistry, anddestined to make even more contributions in other fields in the futures.

To achieve nano-scale resolution and nanofabrication accuracy and toproperly interpret physical and chemical phenomena, it necessary to useatomically flat, atomically smooth substrates over a large areapreferably in the range of several square microns to several squarecentimeters. To produce such substrates, prior art relies ofunsophisticated and inaccurate techniques of attaching an adhesive tapeto the surface of mica or graphite to peel the top most atomic layers toreveal a fresh atomically smooth surface of a piece of mica or graphiteof size and tetchiness. In almost all situations the atomic surface isthe desired result while the lateral shape or size or thickness is oflittle importance. Prior art techniques could not teach methods ofproducing, handling and manipulating samples having a single layergraphite (also called graphene) or mica of a predetermined desirednumber of mono-atomic of mica or graphite.

Graphites are well known and are widely used materials. For example U.S.Pat. No. 6,538,892 exploits its good mechanical and anisotropic thermalproperties for the construction of heat sinks. Graphites according tothe description in U.S. Pat. No. 6,538,892, are made up of layer planesof hexagonal arrays or networks of carbon atoms. These layer planes ofhexagonally arranged carbon atoms are substantially flat and areoriented or ordered so as to be substantially parallel and equidistantto one another, as shown in FIG. 27. The substantially flat, parallelequidistant sheets or layers of carbon atoms, 2710, usually referred toas graphene layers or basal planes, are linked or bonded together andgroups thereof are arranged in crystallites. Highly ordered graphitesconsist of crystallites of considerable size: the crystallites beinghighly aligned or oriented with respect to each other and having wellordered carbon layers. In other words, highly ordered graphites have ahigh degree of preferred crystallite orientation. It should be notedthat graphites possess anisotropic structures and thus exhibit orpossess many properties that are highly directional e.g. thermal andelectrical conductivity and fluid diffusion.

Briefly, graphites may be characterized as laminated structures ofcarbon, that is, structures consisting of superposed layers of carbonatoms joined together by weak van der Waals forces 2712. In consideringthe graphite structure, two axes or directions are usually noted, towith, the “c” axis or direction and the “a” axes or directions. Forsimplicity, the “c” axis or direction may be considered as the directionperpendicular to the carbon layers. The “a” axes or directions may beconsidered as the directions parallel to the carbon layers or thedirections perpendicular to the “c” direction. The graphites suitablefor manufacturing flexible graphite sheets possess a very high degree oforientation.

The bonding forces holding the parallel layers of carbon atoms togetherare only weak van der Waals forces. In a process referred to asexfoliation of graphite, natural graphites can be treated so that thespacing, d, in FIG. 27A between the superposed carbon layers can beappreciably opened up so as to provide a marked expansion of Nd, as inFIG. 27B, the direction perpendicular to the layers, that is, in the “c”direction, and thus forms an expanded graphite structure in which thelaminar character of the carbon layers is substantially retained. Ithas-been shown that N can be in the range of 100 to 1000 according tothe treatment process. The graphite layers are referred to as graphenelayers possess very high electrical and thermal conductivities exceedingthose of copper, while retain high temperatures and exceedingly Youngmodulus.

Recently, Andrei Geim and colleagues of the University of Manchesterisolated a single sheet of graphene and measured its remarkableproperties which include conductivity 100 higher than copper andastonishing Quantum Hall Effect behavior. These and other results aredescribed in January, 2006, Physics Today. These results could be madepossible only after successful isolation of a single 1 Angstrom graphenelayer, a feat that was not previously possible. Geim's team succeeded inisolating a single graphene layer by random and tedious andunpredictable method. According to the Physics Today Article: “Theirmethod is astonishingly simple: Use adhesive tape to peel off weaklybound layers from a graphite crystal and then gently rub those freshlayers against an oxidized silicon surface. The trick was to find therelatively rare monolayer flakes among the macroscopic shavings.Although the flakes are transparent under an optical microscope, thedifferent thicknesses leave telltale interference patterns on the SiO2,much like colored fringes on an oily puddle. The patterns told theresearchers where to hunt for single monolayers using atomic forcemicroscopy.

The work confirmed that graphene is remarkable—stable, chemically inert,and crystalline under ambient conditions.”

From the above and other recent investigations on graphene as well asfrom commercial supplier of graphite substrate, one concludes that thereis a need for inventing convenient, low cost, and fast methods forisolating single layers of graphene and predictable stacks of selectednumber of graphene layers. There is further the need for general methodsfor isolating single layer or predictable number of layers from lamellaror multilayer materials which include but not limited to mica, Superlattices MoS2, NbSe2, Bi2Sr2CaCu2Ox, graphite, mica, Boron nitride,dichalcogenides, trichalcogenides, tetrachalcogenides,pentachalcogenides and Hydrotalcite-like materials.

Therefore, many aspects of the present invention involve production ofsingle and multiple layers of lamellar material. Many of the inventivefeatures and certain embodiments of the present invention rely on theability to make ultra-thin, nano-scale films. In further embodiments, itis desirable that these films are are atomically flat films. Theseenable the fabrication of all the probe configurations that perform avariety of functions necessary to advance the frontier of nano-scienceand technology including but not limited to imaging, analysis,sequencing, nano-lithography, and nano-manipulation as well a variety ofother applications. Thin film deposition methods describe above may beused to produce thing films with Angstrom precision. Alternatively, evenmore precisely define thickness can be produced the controlled peelingof one or more predetermined number of layers from lamellar material astaught herein. These embodiments described herein apply to graphite toproduce graphene layers, to producing layers of mica, MoS2 and lamellarmaterials.

One embodiment to selectively peel off a single layer from a lamellarmaterial, 2810, is illustrated in FIG. 28. The material is cut along theline 2812, at an angle of, for example, 20 degrees or more relative the“c” axis. The goal is the have access to the top most layer 2822, aseach layer is sequentially removed according to FIG. 28B. Two knife edgeprobes, as described herein, having tip opening dimensions small enoughto access individual layers or groups of layers that are revealed due tothe angular cut, are use to facilitate the peeling process. Knife edgeprobe 2818 pushes down on the second layer against the first substrate2814 while knife edge probe 2820, pushes up the first layer against asecond substrate 2816, attached to the desirable first layer. FIG. 28Cshows the complete separation of the first layer that is attached to thesubstrate 2816 which is being pulled vertically to facilitate theseparation process.

In another embodiment, the knife edges 2818, 2820, are applied in thehorizontal directions pushing on both sides pry loose the first layerwhile the substrate 2816 is pulling upward. This method illustrated inFIGS. 28D-F, is facilitated with the knowledge of the exact separationbetween layers by known imaging techniques such as AFM and STM. Thisinformation, along with well know tools to move the knife edges withsub-angstrom precision, allows for reliable separation of the layers.

FIG. 29 illustrates yet another embodiment to reliably separate singlelayers. It exploits etching the peripheral regions of the first layer toexpose the second layer by known etching techniques includingelectrochemical etching as shown in FIG. 29A. Here, a voltage source2918 is applied actoss electrodes 2915, that are contacting theperipheral regions 2910, of the first layer 2922.

The exposed second layer 2912 is pushed as in FIG. 29B. After theetching is complete, the electrodes 2915 push down on the second layeragainst the substrate 2914 while the top substrate 2916 is pullingupward the selected first layer 2922. Thus a single layer isconveniently and inexpensively removed and transferred to a thirdsubstrate, optionally. The substrate 2916 is removably bonded to thefirst layer 2922 by many bonding techniques including but not limited toadhesives, waxes, vacuum, etc. The final result in 29C is repeated forall the other layers of the lamellar material until all layers areremoved with minimum of waste. This method can also be combined withmethod described in FIG. 28A-F above to allow for the selection and theremoval of more than a single layer. For instance, in the graphene case,it may be desirable to have a single layer of 1 Ang, 2 layers of 2 Ang,N layers of multiple Ang, depending on how the graphite is exfoliated toswell the interlayer spacing by factors of 10-1,000. (See exfoliatedgraphite description presented above with respect to FIGS. 27 A and 27B).

Another embodiment that takes advantage of the unique properties ofgraphene and metallically coated other lamellar materials is describedin FIG. 30. A special substrate 3016 is provided and is removableattached to the first layer 3022 we intend to peel. Current source 3012is applied to the first graphene layer 3022 and electrode 3024 depositedon top of substrate 3016. The current 3028 flowing in electrode 3024 andflowing out (in the opposite direction) of single layer 3022 result in amagnetic force 3020 that selectively pulls upward in the upwarddirection 3018, only the first layer 3022. By further applying amechanical force upward to substrate 3016, the combination of magneticand mechanical forces allows peeling with ease 3022. Since no suchforces are influencing second and third layers, they are left intact.Separation process is illustrated in 30A-B.

Instead of exploiting the magnetic force in the aforementionedembodiment, it is possible to use instead electrostatic force adillustrated in 31A-B. In this case a voltage source 3116 is applied toelectrode 3124, deposited on substrate 3112 and a revealed portion ofthe first layer 3122. The electric field 3120 is applied and causes anelectrostatic force in the upward direction 3118, and along with amechanical force applied to a substrate upward in a pulling selection,the first layer is selectively removed from the entire multi layerstructure 3110.

Another embodiment of peeling layers of lamellar material is shown inFIGS. 32A-C. Here the multilayer lamellar structure 3210 is attached toa substrate 3214 to the bottom while at the top implement substrate 3212is removably attached to the top of the specimen. Said substrate 3212may be a vacuum handler, adhesive tapes or other films with removableadhesives. The first step is to lift substrate 3212 which will pull orpeel a random number of layers 3216, shown in FIG. 32A. This process isrepeated as necessary until the last few layers remain as in FIG. 32B.In FIG. 32C the second to last layer is finally removed, leaving thelast layer 3222 bonded to substrate 3214. Note that the shavings, or thepeelings of random number of layers are in turn attached to substrate3214 and the process is repeated until the desired number of singlelayers are removed and utilized.

The above embodiments of methods to selectively remove single layers, orpredetermined number of layers from lamellar could be combined asappropriate to achieve most advantageous, practical and economical wayto produce the desired results.

As discussed herein, in certain embodiments of the herein describedmethods of making the films, the probe tip active area has relevant tipdimensions (e.g., tip width t as shown in the above FIG. 2A) that is afunction of a very thin film that is layered, deposited, or otherwiseformed either on a portion of a probe body or on intermediate structuresbetween plural probes.

As discussed herein, in certain embodiments of the herein describedmethods of making the films, the probe tip active area has relevant tipdimensions (e.g., tip width t as shown in the above FIG. 2A) that is afunction of a very thin film that is layered, deposited, or otherwiseformed either on a portion of a probe body or on intermediate structuresbetween plural probes.

Using various film processing techniques invented by the inventor hereofand incorporated by reference herein above and below, ultra thin layersof materials are deposited to form a stack of layers. The probes areasmay be formed as openings, whereby a series of probes may be readilyformed by creating a stack of layers alternating between insulator orsemiconductor materials and selectively removable materials, whereby thegeometry and dimensions of the selectively removable materials definesthe opening geometry and dimensions. Note the selectively removablematerials may also be placed adjacent a conductor, or between a pair ofconductors, to, e.g., allow for controllable dispensing or otherfunctionality.

In other embodiments, the probes areas may be a suitable conductors,whereby a series of probes may be readily formed by creating a stack oflayers alternating between insulator or semiconductor materials andconductive material, whereby the geometry and dimensions of theconductive material defines the probe or electrode geometry anddimensions.

Certain methods to make the probes, probe sets, and probe arrays mayutilize the processing techniques and various tools invented byapplicants hereof suitable for processing thin layers and formingvertically integrated devices. Various probes and configurations thereofmay be manufactured with the use of Applicant's multi-layeredmanufacturing methods, as described in U.S. Non-provisional applicationSer. No. 09/950,909, filed Sep. 12, 2001 entitled “Thin films andProduction Methods Thereof”; 10/222,439, filed Aug. 15, 2002 entitled“MEMs And Method Of Manufacturing MEMs”; 10/017,186 filed Dec. 7, 2001entitled “Device And Method For Handling Fragile Objects, AndManufacturing Method Thereof”; PCT Application Serial No. PCT/US03/37304filed Nov. 20, 2003 and entitled “Three Dimensional Device Assembly andProduction Methods Thereof”; U.S. Pat. No. 6,857,671 granted on Apr. 5,2005 entitled “Method of Fabricating Vertical Integrated Circuits”; U.S.Non-provisional application Ser. Nos. 10/717,220 filed on Nov. 19, 2003entitled “Method of Fabricating Multi Layer MEMs and MicrofluidicDevices”; 10/719,666 filed on Nov. 20, 2003 entitled “Method and Systemfor Increasing Yield of Vertically Integrated Devices”; 10/719,663 filedon Nov. 20, 2003 entitled “Method of Fabricating Multi Layer Devices onBuried Oxide Layer Substrates”; all of which are incorporated byreference herein. However, other types of semiconductor and/or thin filmprocessing may be employed.

Referring now generally to FIGS. 33A-33F, a method and system for makinga thin device layer 3320 that may be used as a probe or probe precursor,or may be used as a substrate for a probe, probe precursor, probe set,or probe array thereon or therein (generally referred to herein as“probe elements”) according to various embodiments of the presentinvention. FIG. 33A shows a bulk substrate 3302 as a starting materialfor the methods and structures of the present invention. Referring toFIG. 33B, a release inducing layer 3318 is created at a top surface ofthe bulk substrate 3302. This release inducing layer 3318 may include aporous layer or plural porous layers. The release inducing layer 3318may be formed by treating a major surface of the bulk substrate 3302 toform one or more porous layers 3318. Alternatively, the release inducinglayer 3318 in the form of a porous layer or plural porous layers may bederived from transfer of a strained layer to the bulk substrate 3302.

Further, the release inducing layer 3318 may include a strained layerwith a suitable lattice mismatch that is close enough to allow growthyet adds strain at the interface. For example, for a single crystallinesilicon substrate 3302, the release inducing layer in the form of astrained layer may include silicon germanium % other group III-Vcompounds, InGaAs, InAl, indium phosphides, or other lattice mismatchedmaterial that provides for a lattice mismatch that is close enough toallow growth, in ¹For example, U.S. Pat. No. 6,790,747 to SiliconGenesis Corporation, incorporated by reference herein, teaches using asilicon alloy such as silicon germanium or silicon germanium carbon, inthe context of forming SOI; S.O.I.Tec Silicon on Insulator TechnologiesS.A. U.S. Pat. No. 6,953,736, incorporated by reference herein,discloses using a lattice mismatch to form a strainedsilicon-on-insulator structure with weak bonds at intended cleave sites.embodiments where single crystalline material such as silicon is grownas the deice layer 3320, and also provide for enough of a mismatch tofacilitate release while minimizing or eliminating damage to probes orprobe precursors formed in or upon the device layer 3320. The releaseinducing layer 3318 may be formed by treating (e.g., chemical vapordeposition, physical vapor deposition, molecular beam epitaxy plating,and other techniques, which include any combination of these) a majorsurface of the bulk substrate 3302 with suitable materials to form astrained layer 3318 with a lattice mismatch to the device layer 3320(e.g., silicon germanium when the device layer 3320 and the substrate3302 are formed of single crystalline Si). One key feature of therelease layer, particularly in the form of the strained layer, is thatat least a portion of the release layer comprises a crystallinestructure that is lattice mismatched compared to the bulk substrate andthe device layer to be formed or stacked atop the release layer.Alternatively, the release inducing layer 3318 in the form of a strainedlayer may be derived from transfer of a strained layer to the bulksubstrate 3302.

In other preferred embodiments, the release inducing layer comprises alayer having regions of weak bonding and strong bonding (as described indetail in Applicant's copending U.S. patent application Ser. No.09/950,909 filed on Sep. 12, 2001 and U.S. patent application Ser. No.10/970,814 filed on Oct. 21, 2004, both entitled “Thin films andProduction Methods Thereof” incorporated by reference herein, andfurther referenced herein as “the '909 and '814 applications”).

Still further, the release inducing layer may include a layer havingresonant absorbing material (i.e., that absorbs certain excitingfrequencies) integrated therein. For example, when certain excitingfrequencies are impinged on the material such as during debondingoperations, resonant forces cause localized controllable debonding byheating and melting of that material

Referring to FIG. 33C, a device layer 3320 is formed on top of or withinthe release layer 3318. In certain preferred embodiments, the devicelayer 3320 is epitaxially grown, e.g., as an epitaxial single crystalsilicon layer. In still further alternative embodiments, the devicelayer may be attached to the release layer and placed atop the substratelayer or bulk substrate 3302. For example, a suitable vacuum handler(such as one formed as described in 10/017,186 filed Dec. 7, 2001entitled “Device And Method For Handling Fragile Objects, AndManufacturing Method Thereof”, incorporated by reference herein, orother vacuum handlers) may be used to hold and transfer a thin layer asmentioned above.

A buried oxide layer may optionally be provided below the device layer3320. For example, after the step described with respect to FIG. 33B, aportion of the release layer 3318 may be formed into an oxide layer orregion. Alternatively, portions of the release layer 3318 may be treatedto form buried oxide regions. Further, in another example, after thestep described with respect to FIG. 33C, a portion of the release layer3318 may be formed into an oxide layer or region, e.g., with suitableimplantation treatment, or treated to form buried oxide regions. In afurther alternative, where the device layer is attached to the releaselayer, the surface of the device layer intermediate the release layermay be treated to form an oxide layer, or an oxide layer may bedeposited on the surface of the device layer intermediate the releaselayer.

Referring to FIG. 33D, one or more probes and/or probe precursors 3322may be formed in or upon the device layer. In certain embodiments, thedevice layer has wafer scale dimensions, whereby plural probes and/orprobe precursors are formed on the wafer. The release layer 3318 allowsthe device layer 3320 to be sufficiently bonded to the bulk substrate3302 such that during processing of the probes and/or probe precursors3322, overall structural stability remains.

Referring now to FIG. 33F, the device layer 3320 having probes and/orprobe precursors 3322 thereon or therein may easily be separated fromthe bulk substrate 3302. As shown in FIG. 33G, the device layer mayoptionally include a portion 3318′ of the release layer. This may bekept with the device layer, or removed by conventional methods such asselective etching or grinding. This allows one to have a very thindevice layer that may be used alone, e.g., for probes according tocertain embodiments hereof. Alternatively, the thin device layer may bestacked to form a probe (e.g., in the case where the probe precursor isa portion of a probe that is stacked with another probe precursor, forexample, stacked halves of a probe), or to form an array of probes.Further, the remaining substrate 3302 (which may have a portion 3318″ ofthe release layer) remains behind, which may be recycled and reused inthe same or similar process after any necessary polishing.

Accordingly, a method to make thin device layer utilizing the releaselayer described above with respect to FIGS. 33A-33F includes providing astructure A with 3 layers 1A, 2A, 3A, wherein layer 1A is a devicelayer, layer 2A is a release layer, and layer 3A is a support layer. Inthis manner, layer 1A is releasable from layer 3A. One or more probesand/or probe precursors are fabricated on the device layer 1A. Then,device layer 1A may be released from support layer 3A. The support layer3A may be reused for subsequent processes, e.g., as a support layer oras a device layer.

As shown in FIGS. 33A-33F, release layer 3318 may comprise a layer ofporous material, such as porous Si. In a further alternative embodiment,and referring now generally to FIGS. 34A-34G, a method and system formaking a thin layer with a useful device thereon or therein is provided,wherein the release layer comprises a sub-layer 3418 of first porosityP1 and a sub-layer 3426 of second porosity P2. Thus, the release layercomprises a porous release layer having a sub-layer region of relativelylarge pores P1 proximate the substrate and a sub-layer region ofrelatively small pores P2 proximate the device layer. In certainembodiments, sub-layer region P1 is formed directly on said substrate.In other embodiments, sub-layer region P2 is grown on said sub-layerregion P1. Note that although these representations show distinctsub-layers 3418 of first porosity P1 and sub-layers 3426 of secondporosity P2, other porosity gradients across the thickness of theoverall release layer may be used.

FIG. 34A shows a bulk substrate 3402 as a starting material for themethods and structures of the present invention. Referring to FIG. 34B,a porous layer P1 (3418) is created at a top surface of the bulksubstrate 3402.

Referring to FIG. 34C, a second porous layer P2 (3426) may be formed onthe first porous layer P1 (3418). In certain embodiments, a layer 3426may be stacked and bonded to layer 3418. In certain other embodiments, alayer 3426 may be grown or deposited upon layer 3418.

Referring to FIG. 34D, a device layer 3420 is formed on top of theporous layer P2 (3426). In certain embodiments, the device layer 3420 isepitaxially grown, e.g., as a single crystal silicon layer. In stillfurther alternative embodiments, the device layer may be attached to therelease layer, e.g., transferred to the release layer.

A buried oxide layer may optionally be provided below the device layer3420. For example, after the step described with respect to FIG. 34B or34C, a portion of the layer 3418 or 3426 may be formed into an oxidelayer or region. Alternatively, portions of the layer 3418 or 3426 maybe treated for form buried oxide regions. Further, in another example,after the step described with respect to FIG. 34D, a portion of thelayer 3418 or 3426 may be formed into an oxide layer or region, e.g.,with suitable implantation treatment, or portions of the layer 3418 or3426 may be treated to form buried oxide regions. Alternatively, wherethe device layer is attached to the layer 3426, the surface of thedevice layer intermediate the release layer may be treated to form anoxide layer, or an oxide layer may be deposited on the surface of thedevice layer intermediate the release layer.

Referring to FIG. 34E, one or more probes and/or probe precursors 3422may be formed on the device layer. In certain embodiments, the devicelayer has wafer scale dimensions, whereby plural probes and/or probeprecursors are formed on the wafer. The layer 3418 or 3426 allows thedevice layer 3420 to be sufficiently bonded to the bulk substrate 3402such that during processing of the probes and/or probe precursors 3422,overall structural stability remains.

Referring now to FIG. 34F, the device layer 3420 having probes and/orprobe precursors 3422 thereon or therein may easily be separated fromthe bulk substrate 3402. As shown in FIG. 34G, the device layer mayoptionally include a portion 3426 of the porous layer P2. This may bekept with the device layer 3420, or removed by conventional methods suchas selective etching or grinding.

As shown in FIGS. 33A-33F and 34A-34G, release layer 3318 may comprise alayer of strained material, such as a layer of silicon-germanium (SiGe).For example, a layer of SiGe may be grown on a the substrate layer.Since germanium has a larger lattice constant than Si, the SiGe layer iscompressively strained as it grows.

Referring now to FIGS. 35A-35F, another method of making a thin layerincluding one or more probes and/or probe precursors therein or thereonis provided. A bulk substrate 3502 is provided (FIG. 35A). Referring toFIG. 35B, all or a portion of a surface 3504 of the bulk substrate 3502′is treated to form a region 3506. In this embodiment, as describedbelow, region 3506 is formed of a material and/or having materialcharacteristics to allow growth of a layer on top thereof, and alsoserve as a portion of the release layer, wherein portion 3506 representsa weak bond region as described above and described in further detail inApplicant's copending the '909 and '814 applications incorporated byreference herein. In the embodiment shown with respect to FIGS. 35A-35F,a portion of the surface 3504 of the bulk substrate 3502′ is treated,whereby portions 3508 of the surface 3504 remain as the original bulksubstrate which (shown in FIGS. 35B-35F as the periphery, but it is tobe understood that other patterns may be created as described inApplicant's copending the '909 and '814 applications incorporated byreference herein). These portions represent strong bond regions asdescribed in the '909 and '814 applications.

Referring now to FIG. 35C, a single crystalline material layer 3510 suchas single crystalline silicon is epitaxially grown on top of the weakand strong regions 3506, 3508. FIG. 35D shows probes and/or probeprecursors fabricated upon or within the single crystalline materiallayer 3510. Referring to FIG. 35E, portions of the single crystallinematerial layer 3510 are removed corresponding to the regions of theportions 3508, and the portions 3508 are removed, for example bychemical etching, mechanical removal, hydrogen or helium implantationand heating of the portions 3508, or providing a material containing aresonant absorber at the portions 3508 for subsequent heating andmelting of that material. Accordingly, a modified single crystallinematerial layer 3510′ on the portion 3506 remains. FIG. 35F shows theportion 3506 removed, thereby leaving single crystalline material layer3510′ with probe elements 3512 thereon or therein. Alternatively, singlecrystalline material layer 3510′ with probe elements 3512 thereon ortherein may be removed from the portion 3506, for example, by mechanicalcleavage (parallel to the plane of the layers), peeling, or othersuitable mechanical removal, whereby some residue of the portion 3506may remain on the back of the single crystalline material layer 3510′with probe elements 3512 thereon or therein and some residue of theportion 3506 may remain on the top of the bulk substrate 3502″ leftbehind. In this manner, the bulk substrate 3502″ may be recycled andreused with minimal polishing and/or grinding, thereby minimizing wasteof the single crystalline material of the bulk substrate 3502. Thesingle crystalline material layer 3510′ with probe elements 3512 thereonor therein may be used as is, diced into individual devices orstructures, or aligned and stacked (on a probe or probe array scale, oron a wafer scale) to form a probe, probe array, or plurality of probesand/or probe arrays.

In certain embodiments, the strong bond portions 3508 may be formed bystarting with a uniform layer. For example, the surface 3504 maycomprise a strained material, such as silicon germanium. Utilizing zonemelting and sweeping techniques, the germanium swept away from thedesired strong bond regions 3508. When a layer 3510 is grown or formedon the layer having portions 3506, 3508, layer 3510 will be stronglybonded at the regions of portions 3508 and relatively weakly bonded atthe regions of portions 3506.

Referring now to FIGS. 36A-36F, another method of making a thin layerincluding one or more useful devices or structures therein or thereon isprovided. A bulk substrate 3602 is provided (FIG. 36A). Referring toFIG. 36B, all or a portion of a surface 3604 of the bulk substrate 3602′is treated to form porous sub-regions 3605 and 3606. In this embodiment,as described below, region 3606 is formed of a material and/or havingmaterial characteristics to allow growth of a layer on top thereof, andalso serve as a portion of the release layer, wherein porous sub-regions3606/3605 represent a weak bond region as described above and describedin further detail in the '909 and '814 applications incorporated byreference herein. In the embodiment shown with respect to FIGS. 36A-36F,a portion of the surface 3604 of the bulk substrate 3602′ is treated(forming sub-regions 3605/3606), whereby portions 3608 of the surface3604 remain as the original bulk substrate which (shown in FIGS. 36B-36Fas the periphery, but it is to be understood that other patterns may becreated as described in Applicant's copending the '909 and '814applications incorporated by reference herein). These portions representstrong bond regions as described in the '909 and '814 applications.

Thus, the release layer comprises sub-regions 3605/3606 and portions3608. Sub-region 3605 has relatively large pores P1 proximate thesubstrate and sub-region 3606 has of relatively small pores P2 proximatethe device layer to be described below. In certain embodiments,sub-region 3605 is formed directly on said substrate, and sub-region3606 is grown on said sub-region 3605. In certain embodiments,sub-region 3606 may be stacked and bonded to sub-region 3605. In certainother embodiments, sub-region 3606 may be grown or deposited uponsub-region 3605.

Referring now to FIG. 36C, a single crystalline material layer 3610 suchas single crystalline silicon is epitaxially grown on top of the weakand strong regions 3606, 3608. FIG. 36D shows devices or structuresfabricated upon or within the single crystalline material layer 3610.Referring to FIG. 36E, portions of the single crystalline material layer3610 are removed corresponding to the regions of the portions 3608, andthe portions 3608 are removed, for example by chemical etching,mechanical removal, hydrogen or helium implantation and heating of theportions 3608, or providing a material containing a resonant absorber atthe portions 3608 for subsequent heating and melting of that material.Accordingly, we are left with a modified single crystalline materiallayer 3610′ on the portion 3606. FIG. 36E shows an exemplary cleavingdevice, for example a knife edge device, water jet, or other device,used to cut between the sub-regions 3605 and 3606. FIG. 36F shows thebottom portion of sub-region 3606 removed (with a portion of sub-region3606 remaining on the bottom of the single crystalline material layer3610), and the top portion of sub-region 3605 removed (with a portion ofsub-region 3605 remaining on the bulk substrate 3602″). Accordingly, thesingle crystalline material layer 3610′ is left with devices orstructures 3612 thereon or therein. In this manner, the bulk substrate3602″ may be recycled and reused with minimal polishing and/or grinding,thereby minimizing waste of the single crystalline material of the bulksubstrate 3602. The single crystalline material layer 3610′ with devicesor structures 3612 thereon or therein may be used as is, diced intoindividual devices or structures, or aligned and stacked (on a device orstructure scale, or on a wafer scale) to form a vertically integrateddevice.

Referring to FIG. 37, a starting multiple layered substrate 3700 isshown. The substrate 3700 may be, in certain preferred embodiments, awafer for processing thousands or even millions of probe elements, or beused to derive a very thin layers for use as probes and/or probeprecursors.

The multiple layered substrate 3700 includes a first device layer 3710selectively bonded to a second substrate layer 3720, having stronglybonded regions 3703 and weakly bonded regions 3704. Using the techniquesdescribed in the above-mentioned patent applications, or other suitablewafer processing and handling techniques, the first layer 3710, intendedfor having one or more probe elements therein or therein, or used as aprobe or probe precursor as a very thin layer, may readily be removedfrom the second substrate layer 3720 (which serves as mechanical supportduring device processing) with little or no damage to the structure(s)formed (including material deposited or otherwise incorporated, or wellsor other subtractions to the layer 3710) in or on the device layer 3710.

Accordingly, according to the methods of FIGS. 33 and 34, a layeredstructure is formed generally includes a first layer suitable for havinga useful element formed therein or thereon releasably attached or bondedto a second layer, e.g., a substrate. A method to form a layeredstructure generally comprises releasably adhering a first layer to asecond layer. Further, according to the methods of FIGS. 35-37, alayered structure is formed generally includes a first layer suitablefor having a useful element formed therein or thereon selectivelyattached or bonded to a second layer, e.g., a substrate, with regions ofweak bonding and regions of strong bonding. The layered structure may beused for production of various devices including probes and/or probeprecursors as provided for herein. Alternatively, a layered structuremay be used as a source of one or more probes and/or probe precursors,for example, when the device layer is used as the probe, whereby thecapability to produce and remove with little or no damage allows forultra thin layers that may be used for ultra high resolution probes.

The separation, for example, shown at steps of FIGS. 33E, 34F, 35E and36E, may comprise various separation techniques. These separationtechniques includes those described in further detail in Applicant'scopending the '909 and '814 applications, incorporated by referenceherein. The separation may be multi-step, for example, chemical etchingparallel to the layers followed by knife edge separation. The separationstep or steps may include mechanical separation techniques such aspeeling, cleavage propagation; knife edge separation, water jetseparation, ultrasound separation or other suitable mechanicalseparation techniques. Further, the separation step or steps may be bychemical techniques, such as chemical etching parallel to the layers;chemical etching normal to the layers; or other suitable chemicaltechniques. Still further, the separation step or steps may include ionimplantation and expansion to cause layer separation.

The material for the layers used herein, as the device layer, therelease layer and the substrate layer, may be the same or differentmaterials, and may include materials including, but not limited to, anyof the lamellar materials described above, plastic (e.g.,polycarbonate), metal, semiconductor, insulator, monocrystalline,amorphous, noncrystalline, biological (e.g., DNA based films) or acombination comprising at least one of the foregoing types of materials.

Further, the release layer may comprise a material layer having certainamounts of dopants that excite at known resonances. When the resonanceis excited, the material may locally be heated thereby melting the areassurrounding the dopants. This type of release layer may be used whenprocessing a variety of materials, including organic materials andinorganic materials.

The device layer and the substrate layer may be derived from varioussources, including thin films described herein, wafers or fluid materialdeposited to form films and/or substrate structures. Where the startingmaterial is in the form of a wafer, any conventional process may be usedto derive the device layer and/or the substrate layer. For example, thesubstrate layer may consist of a wafer, and the device layer maycomprise a portion of the same or different wafer. The portion of thewafer constituting the device layer may be derived from mechanicalthinning (e.g., mechanical grinding, cutting, polishing;chemical-mechanical polishing; polish-stop; or combinations including atleast one of the foregoing), cleavage propagation, ion implantationfollowed by mechanical separation (e.g., cleavage propagation, normal tothe plane of the layers, parallel to the plane of the layers, in apeeling direction, or a combination thereof), ion implantation followedby heat, light, and/or pressure induced layer splitting), chemicaletching, or the like. Further, either or both the device layer and thesubstrate layer may be deposited or grown, for example by chemical vapordeposition, epitaxial growth methods, or the like.

The dimensions of the device layers may also vary in thickness andsurface area. For example, fabrication of probes having ultra highresolution may benefit from the methods and embodiments herein, wherebyprobes may be formed on layers that are a few tenths of a nanometer to afew nanometers.

The surface areas for the methods and embodiments of the presentinvention may be die-scale, wafer scale, or in larger sheets;accordingly, surface areas may be on the order of nanometer(s) squaredto a few microns squared for die-scale; on the order of a centimeterssquared for wafer-scale; and on the order of centimeters squared to ameters squared for sheet scale.

Referring now to FIGS. 38A and 38B, top isometric and sectional views,respectively, are provided of a selectively bonded substrate 3800 havinga plurality of wells 3830 formed in the weakly bonded regions of theselectively bonded substrate 3800. The wells may be formed by etching,mechanical subtraction methods, ion or particle beam etching, or othersuitable methods. Note that the pattern of weak bond regions and strongbond regions may vary, as described in the '909 and '814 applications.However, in certain preferred embodiments, all of the wells 3830 areformed at the weak bond regions of the device layer 3810 and supportedduring processing by the support layer 3820.

FIGS. 38C and 38D show plan and sectional views, respectively, of asingle well 3830 formed in the device layer 3810 described above.Referring to FIG. 38C, the intersecting region between the dashed linesand the walls 3832 of the wells 3830 shows regions wherein probeelements may be processed in certain embodiments, as describedhereinafter. In other embodiments, there may be only one intended regionfor processing nozzles (e.g., on the left or right sides as shown inFIGS. 38C and 38D).

In further embodiments, the wells may be formed only at the intendedprobe element region, e.g., resembling grooves having the thicknessshown by the dashed lines.

Referring also to FIG. 39, the well 3830 generally has angular walls3832, the function of which will be readily apparent. Further, thecenter recessed portion 3834 of the well will become part of a reservoirof the probes. At the top surface of the device layer 3810 adjacent theouter ends of the angular walls 3832 are plateau regions, whichultimately may be part of the inside wall of the probes as describedherein.

Referring now to FIG. 39, a layer 3810 (e.g., having thickness on theorder of about 0.1 nanometers to about 10 nanometers, 10 nanometers toabout 100 nanometers, or 100 nanometers to 1000 nanometers) isselectively bonded to a support layer 3820 as described with respect toFIGS. 33-37 and in the '909 and '814 applications. A region of reservoir3830 is etched away or otherwise removed from a region of the devicelayer in the weak bond region 3803. Suitable nano-scale materialsubtraction methods may be used.

Referring now to FIG. 40A, a layer 3838 (e.g., having thickness on theorder of about 0.1 nanometers to about 10 nanometers, 10 nanometers toabout 100 nanometers, or 100 nanometers to 1000 nanometers) of material,preferably material that is easily removable by etching or othersubtractive methods, is deposited on the wafer. This material may beconductive, such as copper, silicon oxide, aluminum, or other suitablematerials. This space will later become the opening for the nozzle.

Referring to FIG. 40B, a fill material 3840 may optionally beincorporated, also of easily removable material in certain embodiments.The material optionally used to fill the wells during processing andstacking may be the same or different from the material used at theplateaus (that will form nozzle walls).

In certain embodiments, since the device layer including the etched wellhaving suitable material deposited thereon is generally positioned overthe weak bond region 3803 of the multiple layered substrate 3800, thedevice layer 3810 may readily be removed from the support layer 3820.For example, the strong bond regions 3804 may be etched away by throughetching (e.g., normal to the surface through the thickness of the devicelayer in the vicinity of the strong bond region), edge etching (parallelto the surface of the layers), ion implantation (preferably withsuitable masking of the etched well and deposited material to form thenozzle, or by selective ion implantation), or other known techniques.Since the above techniques are generally performed at the strong bondregions 3804 only, the etched well and material deposited in the weakbond regions 3803 are easily released form the substrate, asschematically shown in FIG. 41, for example with a handler 3850.

Referring now to FIG. 42, several layers 3810 including etched wells3830 having material deposited 3838 thereon (and optionally fill 3840)may be stacked to form a structure 3860. The structure 3860 may furtherinclude a solid layer 3862, e.g., to form a wall for the top-most nozzleas shown in FIG. 42. Although in certain embodiments precise alignmentmay be desired at this point, certain embodiments may use relaxedalignment standards at this point, as will be apparent from the furtherdescribed steps.

As shown in FIG. 43, the wafer stack 3860 can now be sliced along a cutline 3864, creating two portions with exposed reservoirs. From theopposing side, these devices can also be sliced along the line 3866. Theend may be grinded and polished until it is very close to the etchedaway reservoir, but no less than the desired nozzle length.

Referring now to FIGS. 44 and 45, the deposited material 3838 may beetched away, exposing an etched channel 3868 (e.g., 5 nm opening whenthe material deposition layer is 5 nm). A material reservoir 3870 (orregion 3870 for other purposes, depending on the desired use of thenozzle structure) remains behind the opening 3868. Each etched channel3868 is generally spaced apart by approximately the thickness of thedevice layer 3810. Thus, a nozzle device 10 having plural openings 3868each associated with regions 3870 is provided. Accordingly, when thethickness of the material to be removed is extremely small, e.g., on theorder of about 0.1 nanometers to about 10 nanometers, 10 nanometers toabout 100 nanometers, or 100 nanometers to 1000 nanometers, the extendededge probe tip as described above is created at the openings 3868.

Alternatively, and referring to FIG. 46, to form an opening less thanthe width of the entire edge, the outside portions may be masked 3872prior to etching the deposited material 3838 to form openings 3868′.Thus, a nozzle device 3810′ having plural openings 3868′ is provided.Accordingly, the width (i.e., the y direction as shown in FIGS. 44-46)of the probes may be the same or different from the width of the wells.In certain embodiments, it may be desirable to provide wells havingwidths larger than that of the nozzle to increase the material capacityof the well while maintaining the nozzle dimensions as small aspossible.

In a further embodiment, and referring now to FIGS. 47 and 48, a nozzledevice 3880 (e.g., as describe herein), of a single layer, may berotated approximately 90° with respect to the stack of layers 3860having layers 3838 deposited therein at the locations of the nozzles.Etchant may be filled in the reservoir of the rotated nozzle structure3880, and the openings 3882 of the nozzles may be formed. Using thistechnique, it is possible to create nozzles having approximately thesame width and height with extremely small dimensions as provided forherein. Thus, a nozzle device 3810″ having plural openings 3868″ isprovided.

Referring now to FIGS. 49 and 50, another embodiment of a method offorming very small width nozzle diameters. As described with referenceto FIGS. 44 and 45, the deposited material between layers may be etchedaway, exposing an etched channel spaced apart by approximately thethickness of the device layer.

These etched channels 3868 may then be filled with an etchable material.For example, a nozzle device 3880 as describe herein, of a single layer,may be rotated approximately 90° with respect to the stack of layershaving material etched away at the locations of the nozzles. An etchablematerial may be filled in the reservoir of the rotated nozzle structure,which is filled at the regions where the nozzles on the stack of layersare to be formed. The surrounding areas between the layers are thenfilled with a plug material. Then the etchable material in the nozzleregion is etched away, exposing the nozzles 3868′″. Using thistechnique, it is possible to create nozzles having approximately thesame width and height of extremely small dimensions. Thus, a nozzledevice 3810′″ having plural openings 3868′ is provided.

Note this etchable material should be selectively removable by anetchant (e.g., not removing the bulk material).

Referring now to FIGS. 51A and 51B, a nozzle array 5100 of the presentinvention is shown. Therein, one or more spacer layers 5102 may bepositioned between a desired number of to-be-formed channels, e.g.,during stacking of the well structures.

Referring to FIG. 52, an enlarged cross section of stacked layers usedto form the probes such as nano-probes having wells and tip portionswith tip active area dimensions equal or less than the sub-objects beinganalyzed by the specimen, or of a nanometer or sub-nanometer scale forother applications as described herein. These tip portions are alsoformed to desired tip length, is shown. As described above, the layers3838 have been processed to form the wells 3830 and nozzle tip regionsgenerally by deposition of a layer 3838 of material capable of beingselectively removed (e.g., etched) therein (the well) and thereon (theshelf at the top of the well), as described herein. The materialscapable of being selectively removed for the plateau and/or the well maybe the same or different. The wells and plateaus have various dimensionsthat will characterize the nozzle array ultimately formed. The nozzlehas a tip length N_(L), a tip opening height N_(O), and a period P.

Note that the dimensions of such nozzles may be on the order of a lessthan a nanometer (e.g., less than 0.1 nm) to 10 or 10 s of nanometers,on the order of 10 or 10 s of nanometers to 100 or 100 s of nanometers,or on the order of a tenth of a microns or tenths of a micron to amicron or a few microns, depending on the desired application. Further,the arrays may be spaced apart by a few nanometers to several microsapart.

Referring to FIG. 53, an enlarged cross section of stacked layers usedto form the micro and nano nozzles herein is shown, detailing grindstops 5386 provided to enhance the ability to control the nozzle lengthNL. In certain embodiments, it is desirable to minimize the nozzlelength. A grind stop 5386 is provided proximate the desired nozzlelength. The grind stop may be provided during processing of the wells onthe device layer. Further, the grind stops may further serve asalignment marks, e.g., as described in aforementioned U.S. patentapplication Ser. No. 10/717,220, incorporated by reference herein.

Referring to FIGS. 54A and 54B, an enlarged cross section of stackedlayers used to form the micro and nano probes, and a front view of theopen tip of the prove, respectively, are shown. Note that in certainembodiments, the well 5470 has a width (y direction) greater than thatof the nozzle tip 5468.

Note that in any of the herein described probe elements, associatedstructures may be provided. For example, in certain embodiments, one ormore electrodes may be provided to facilitate material discharge,detection capabilities, etc. Further, one or more processors, micro ornano fluidic devices, micro or nano electromechanical devices, or anycombination including the foregoing devices may be incorporated in anozzle device. In certain preferred embodiments, electrodes are providedat the nozzle openings and/or wells, and an electrode controller and/ora microfluidic device (e.g., to feed or remove material from thenozzles) is associated with an array of nozzles.

Further, and referring now to FIGS. 55A-D, an exemplary method of makingprobes with open tips and having various conductors (e.g., serving aselectrodes) within an open region in the body for the probe is depicted.FIG. 55A shows a starting section of a multiple layer substrate withlayers 5510 and 5520 as described hereinabove. An well 5530 generallyhas angular walls 5532 and a center recessed portion 5534, althoughother shapes may be provided. Plateau regions 5536 form the openingwalls or supports.

A layer 5538 of conductive material is deposited on the wafer. Aremovable fill material 5540 may be provided in the well to facilitatelayering. Referring to FIG. 55B, a removable fill layer 5542 is providedon the surface having the conductive layer 5538 and the optionally fillmaterial 5540. In this embodiment, the opening of the probe will beformed at the fill layer 5542. Further, a conductive layer 5544 isdeposited or layered on the fill layer 5542, forming a nozzlesub-structure 5550.

Referring now to FIG. 55C, a plurality of nozzle sub-structures 5550 arealigned and stacked (e.g., as described above with respect to FIG. 42).Referring to FIG. 55D, nozzle openings 5560 may be formed, e.g.,according to one of the methods described above with respect to FIGS.44-50, or other lithography or oxidation methods. Note that the plugmaterial may be conductive or insulating, depending on the desiredproperties of the probe.

Referring now to FIG. 56, an enlarged view of a nozzle structure 5600 isprovided, viewing a nozzle opening 5602. Nozzle opening 5602 isgenerally positioned on a nozzle layer “N” between a top portion “A” anda bottom portion “B” (although top and bottom are considered to berelevant for the purpose of description herein only). To describevarious embodiments of possible configurations, sections N, A and B havebeen divided into descriptive sections. These descriptive sections maybe actual discrete regions of different material, or in certainembodiments multiple descriptive sections may be of the same materialand thus actually a uniform region, as will be apparent from the variousembodiments herein.

AA and BB may be the same or different materials, such as insulator orsemiconductor materials to provide the structure of the nozzle 200,electrically insulate the nozzle openings from one another, fluidly sealthe openings from one another, or other functionality.

In certain embodiments, the descriptive sections AL, AC, AR, NL, NR, BL,BC and BR are all of the same materials as AA and BB.

Any combination of AL, AC, AR, NL, NR, BL, BC and/or BR may be providedin the form of conductors. For example, referring back to FIG. 46, uponremoval of the mask after etching the nozzle opening, a structure may beprovided having AL, AC, AR, BL, BC and BR of the same materials as AAand BB, and NL, NR of conductive material.

Further, one or more conductors (e.g., electrodes) may be includedinside within the probes, thereby enabling creation of fields across thenozzle opening. For example, NL and NR, AC and BC, AL and BR, AR and BL,AL, AR and BL, BR may all be electrode pairs to provide any desiredfunctionality. Additionally, one or more conductive electrodes may bewithin the well regions, e.g., to provide electromotive forces to movematerials.

Referring now to FIGS. 57A-C, an example of a method of manufacturingthe herein described nozzles is shown whereby a plurality of sub-layers5702 form each layer 5710. Wells 5730 are processed through the layer5710 as shown in FIG. 57B. FIG. 57C shows nozzle openings 5760 havingplural sub-layers 5702 therearound. These sub-layers may be very useful,for example, where precise metrology is desired.

For example, in certain embodiments, the sub-layers 5702 are formed tovery precise tolerances, e.g., having thicknesses on the order of 0.1 toabout 5 nanometers. When these sub-layers 5702 are formed of differingmaterials (e.g., alternating between insulator and semiconductor,semiconductor and conductor, or conductor and insulator), precise stepmotion may be enabled in the nozzle structures based on known dimensionsof the nozzle sub-layers.

While is possible to use conventional lithographic tools such aselectron beams, particle beams, UV, X-ray, etc., to define certainfeatures herein, extending them to the nano-scale becomes verycumbersome and expensive. In the present invention, certain embodimentsmay benefit from the use of applicants nanolithography tools describedin applicants U.S. patent application Ser. No. 11/077,542 filed on Mar.10, 2005 and entitled “Nanolithography and Microlithography Devices andMethod of Manufacturing Such Devices” incorporated by reference herein.This is advantageous in that a company, easy to use and inexpensive toolmay be proved. Further, use of applicants nanolithography toolsdescribed in above referenced U.S. patent application Ser. No.11/077,542 may advantageously provide extremely small future sizes downto angstrom scale.

Various probes and configurations thereof may be manufactured with theuse of Applicant's microlithography and nanolithography tools andmethods, as described in U.S. Non-provisional application Ser. No.11/077,542 filed on Mar. 10, 2005 entitled “Nanolithography andMicrolithography Devices and Method of Manufacturing Such Devices”.

In certain embodiments herein, a probe may be formed by folding a verythin layer to expose a point at the outside of the fold angle, therebycreating a probe tip with a very small active area suitable for thevarious applications provided for herein including ultra high resolutionanalyses of the specimen at the sub-object level (e.g., nucleotide levelof a DNA or RNA strand or fragment).

For example, and referring now to FIGS. 58-60, a method of manufacturinga probe 5802 is shown. FIG. 58A shows an ultra thin layer 5804 bonded toa first surface 5808 of a base layer 5806. The base layer 5806 maycomprise any suitable material, for example, that will form a portion ofthe probe body, or that may be further processed for additional featuresand/or functionality. The ultra-thin layer 5804 may comprise anysuitable material that may be deposited, laminated or otherwise formedon the surface 5808 of base layer 5806.

Referring now to FIG. 58B, a well 5812 of suitable geometry is etched orotherwise created on surface 5810 of base layer 5806. In certainembodiments, it may be desirable to configure the well such that thedeepest portion is very close to the thin layer 5804. In otherembodiments, it may be desirable to configure the well such that thedeepest portion exposes the back surface (i.e., the surface attached tosurface 5808 of base layer 5806) of the thin layer 5804.

Referring now to FIG. 58C, surface 5810 may optionally be coated with abending layer 5814 formed of a material that has flexiblecharacteristics, including but not limited to polyvinyl alcohol,silicone, or other suitable flexible and stretchable polymeric or othermaterials.

Referring now to FIG. 58D, the composite of layer 5804 and base layer5806 is folded to diverge opposing angled portions of the well 5812.Folding is completed to provide a probe precursor structure 5802′, shownin FIGS. 59A and 59B. As shown, the probe precursor structure 5802′ hasa cross section in a substantially pentagonal shape resembling atriangle adjacent a rectilinear polygon. Of course, one may alter thisshape by changing the shape of the well 5812. Further, the shape may besymmetrical as shown, or asymmetrical. One of the benefits of thepresent folding techniques is that certain alignment requirements may berelaxed.

The bending layer 5814 may be removed. Further, to expose the probe tipactive area 5820, the tip edge 5816 of the structure 5802′ may begrinded, polished, or otherwise removed to expose the folded thin layerof material. The dimension of the probe tip active area 5820 is definedby a multiple of the thickness of the layer 5804, in this case 2 t.

Notably, with the methods of making and manipulating thin films asdescribed above, extremely small tip dimensions for the probe tip activearea are possible. For example, if the layer 5804 is a single twodimensional layer of graphene, then the tip dimension 2 t as shown inFIG. 60 may be on the order of 2 angstroms, and is highly conductive.

Alternatively, the layer 5804 may be formed of a material that can beselectively removed (either completely or partially) to open a channelor path. Nonetheless, in either embodiment, the tip dimensions for thetip active area 5820 are a multiple of the thickness of the layer 5804deposited, layered, or otherwise formed on the base layer 5806.

In another embodiment, and referring now to FIGS. 61A-61J, methods ofmanufacturing a probe 6102, 6102′ or 6102″ are shown. FIGS. 61A and 61Bshow a base layer 6106 having a well 6112 of suitable geometry is etchedor otherwise created on surface 6110 of base layer 6106. In certainembodiments, it may be desirable to configure the well such that thedeepest portion is very close to the thin layer described below. Inother embodiments, it may be desirable to configure the well such thatthe deepest portion exposes the back surface (i.e., the surface attachedto surface 6108 of base layer 6106) of the thin layer described below.

The base layer 6106 may comprise any suitable material, for example,that will form a portion of the probe body, or that may be furtherprocessed for additional features and/or functionality.

Referring now to FIG. 61C, portions 6124 are removed from the base layer6106, generally from the side of surface 6108. Referring now to FIG.61D, portions 6124 are filled with a suitable material 6126. Thismaterial 6126 may be an insulating or conducting plug material (if aprobe in the configuration of FIG. 61H or 61I is desired), or thematerial 6126 may comprise a removable substance (if a probe in theconfiguration of FIG. 61J is desired).

Referring now to FIG. 61E, an ultra thin layer 6104 bonded to thesurface 6108 of base layer 6106 having material portions 6126 to form aflat surface. Known techniques may be applied to smooth the surfaceformed by both the surface 6108 of base layer 6106 having materialportions 6126. Alternatively, the methods for forming atomically smoothsurfaces described herein may be employed.

The ultra-thin layer 5804 may comprise any suitable material that may bedeposited, laminated or otherwise formed on the surface 5808 of baselayer 5806. In certain preferred embodiments, thin films formedaccording to the embodiments herein are used.

Notably, with the methods of making and manipulating thin films asdescribed above, extremely small tip dimensions for the probe tip activearea are possible. For example, if the layer 6104 is a single twodimensional layer of graphene, then the tip dimension is 2 t as shownabove in FIG. 60.

Alternatively, the layer 6104 may be formed of a material that can beselectively removed (either completely or partially) to open a channelor path. Nonetheless, in either embodiment, the tip dimensions for thetip active area 6120 are a function of the thickness of the layer 6104deposited, layered, or otherwise formed on the base layer 6106.

Referring now to FIG. 61F, surface 6110 may optionally be coated with abending layer 6114 formed of a material that has flexiblecharacteristics, including but not limited to polyvinyl alcohol or othersuitable polymeric or flexible metallic material.

The composite of layer 6104 and base layer 6106 having material portions6126 is folded to diverge opposing angled portions of the well asdescribed above with respect to FIG. 58. A probe or probe precursorstructure 6102 is provided as shown in FIG. 61H (after the material ofthe optional bending layer 6114 is removed as shown in FIG. 61G). If aprobe 6102′ is desired, the selectively removable material is used asthe material for layer 6104, and may be removed at this stage, whereby agap 6128. Further, if probe 6102″ is desired, the selectively removablematerial is used as material 6126 and may be removed at this stage,whereby a cavity 6140 is created.

Referring to FIGS. 62A-62B, note that cavities of various configurationsand dimensions 6240′, 6240″ may readily be created by varying theconfigurations and dimensions of portions 6124 described above withreference to FIGS. 61A-61J.

Referring now to FIG. 63A-63D, another alternative method of makingvarious probes with additional versatility and functionality accordingto the present invention is provided. In this case, the structure havinga thin layer 6304 on a base layer 6306 with a well 6312 at the surfaceopposite the thin layer 6304 is folded so that angled portions of thewell 6312 converge as shown. The bending layer or material 6314 may beremoved, resulting in probe 6302 having a tip 6340.

Referring now to FIG. 64, a probe 6410 as formed by various aspects ofthis invention may be utilized to assist in the folding. For example,probe 6410 may be used to contact within the well, whereby themechanical forces assist in the folding processes. In furtherembodiments, vacuum suction may be applied through the probe 6410 toassist in the folding processes.

Referring now to FIG. 65, a plurality of probes 5802 may be aligned andstacked, for example, by stacking edgewise on a platform 6530 andaligning by stacking the tips of the probes 5802 adjacent an alignmentdevice 6534, or stacking the probes 5802 and displacing misalignedprobes 5802 by pushing them into alignment with the alignment device6534, thereby forming probe sets or probe arrays. In certain preferredembodiment, alignment device 6534 has a surface that contacts the tipsand provides for sub-angstrom resolution motion to precisely displaceand align the tips of the probes in an array or probe set. It isparticularly advantageous if the surface that contacts the tips isatomically flat and smooth, for example, as may be produced by variousmethods described herein.

Referring now to FIGS. 66A-66D and FIGS. 67A-67E, another method offorming probes according to the present invention is shown, particularlyopen tip probes. In particular, the tip opening dimensions t are definedby use of spacers such as particles, tubes, spheres, molecules, or otherstructures having precisely defined heights when disposed on asubstrate. These spacers may have extremely small defined dimensions(e.g., a diameter of a sphere or tube that provides the height), such asin the ranges of 0.1 nanometers to about 10 nanometers, 10 nanometers to100 nanometers, and 100 nanometers to 1000 nanometers.

In one example, and referring to FIG. 66A-66D, a plurality of spacers6614 are disposed generally in an orderly fashion upon the surface of asubstrate 6610. As shown in FIG. 66A, the spacers 6614 may, for example,be aligned in groups along the x direction and spaced apart from oneanother in the y direction. Alternatively, the spacers 6614 may be in acontiguous form.

Referring now to FIG. 66B, a superstrate 6620 is provided on the spacers6614 to complete the probe or probe precursor by defining an opening6624. Alternatively, and referring to FIGS. 66C and 66D, the probe maybe cut into segments as shown by dashed lines in FIG. 66C.

In another example, and referring to FIGS. 67A-67B, a plurality ofspacers 6714 are disposed generally in a random fashion upon the surfaceof a substrate 6710. Referring now to FIG. 67B, a superstrate 6720 isprovided on the spacers 6714 to complete the probe or probe precursor bydefining an opening 6724.

Referring now to FIG. 1, a schematic overview of a system of the presentinvention for analyzing extended object specimens is shown. The system100 generally includes a specimen platform 128, a probe set 130 and adetector sub-system 132. The platform 128 is operably coupled to amotion controller 138, for controlling motion of the platform.Alternatively, the specimen may be moved within the platform. In afurther alternative, the probe set (and optionally the associateddetector sub-system) may be moved relative to the platform with thespecimen. Further, the system 100 includes a bias sub-system 136 forcontrol of field application (voltage applied across base and probe) andoptionally other stimuli. In general, in certain systems describedherein, when a hybridization event occurs, a measurable increase incurrent is detected.

In certain embodiments, a low detection voltage may be applied in aconstant manner across the probe set and the platform. However, biasedvoltage application may be utilized to minimize or eliminate noise.

Data regarding the specimens is collected and processed by a processorsub-system 134, which is coupled to an output sub-system (e.g., adisplay, data port, etc.) 140.

In operation, a specimen such as a single stranded polymer (e.g., adenatured strand of DNA) is directed through a path or channel in theplatform. The probe set detects characteristic features of the polymerspecimen, preferably detecting characteristic about each sequentialmonomer in the specimen polymer. The specimen is moved relative theprobe set in a controlled manner, e.g., by step motion to allow theprobe set to obtain characteristic information about each monomer orgroup of monomers. The sequence information is collected, processed andoutputted.

In certain embodiments, high resolution is attainted by utilizing aprobe having a tip dimension, or an active tip area, that is equal to orless than a characteristic sub-object of the extended object, such as anucleic acid within a DNA or RNA strand or fragment. In furtherembodiments, the width dimension of the probe is much larger than thewidth of thickness of the extended object, for example, having width wof about 10 nanometers to about 100 nanometers, 100 nanometers to 1000nanometers, or several microns for analyzing specimens such as typicalDNA strands or fragments. Further, the enlarged width dimension ascompared to the tip or active area is useful in that additionaltolerance is provided for the path of channel of the specimen and/or thestretching procedures.

Referring now to FIG. 68, an embodiment of an ultra-fast DNA sequencingsystem 6800 is shown. The sequencing system uses a nozzle array 6810, asdescribed herein. Further, the sequencing system uses a nano-metrologysystem 6820 to precisely guide denatured DNA strands across theindividual nozzles in the nozzle array.

Referring now to FIG. 69, a schematic of major components of theultra-fast DNA sequencing system 6800 are shown. A nano-nozzle set arrayplatform 6830 upon an N-channel specimen array platform 6828 is operablyconnected to a detector array 6832 associated with a processor 6834,generally for determining instances of hybridization events induced bythe biases applied via a gated bias array control 6836. The DNAspecimens are maintained and displaced in relation to the array with astepped motion control 6838, which is also operably connected to theprocessor 6834. The array platform 6828 is movable at a velocity ofabout 0.1 to about 1 cm/s. Preferably, as shown, the motion is in astepped manner, as described herein. The sequencing results are shown ona sequence display 6840.

The stepped motion is important in preferred embodiments, as the motionand number of steps helps maintain knowledge of position on the ssDNA,and ultimately the position of hybridization events. The stepped motionmay be from about 5% to about 100% of the nozzle opening dimension,preferably about 10% to about 25% of the nozzle opening dimension.

The gating is also important in preferred embodiments, as extremelysynchronized current measurements, bias, motion steps, or otherexcitations are crucial to ultra-fast real time DNA sequencing.

Referring now to FIG. 70, a top view of the ultra-fast DNA sequencingsystem 6800 is shown. The DNA specimens are denatured and maintainedwithin channels 6844.

Referring now to FIGS. 71A-B (wherein FIG. 22A is a section along lineA-A of FIG. 70), each channel 6844 includes biasing systems for applyingvoltages across the DNA samples. As described in more detail herein,hybridization events induce measurable current variations across each ofthe nanonozzles within the nanonozzle set array platform. Preferably,the alignment between the nanonozzles and the channels is extremelyprecise.

Referring now to FIGS. 72A-C, a system 7200 including series of probesets 7230, a probe set 7230 including nozzles or probes 7242, 7244, 7246and 7248, and an enlarged view of probe 7248 are shown. The nanonozzleset array platform 7200 includes nanonozzles with wells, or nucleotidereservoirs, of A, C, T and G molecules. The strands are moved along thechannel and molecules from the nucleotide reservoirs interact with themolecules of the strand through the nozzle. These molecules hybridizewith one other molecule (e.g., A with T, C with G). In general, thehybridization event (e.g., as shown in FIG. 72C) produces measurable anddetectable current pulses, thereby allowing identification of themolecule.

Referring now to FIG. 73, detailed views of hybridization events areshown. In certain detection schemes described herein, a hybridizationevent at the nanonozzle results in a measurable current pulse.

Referring now to FIG. 74, it is shown that, of all possible 16combinations of A, T, G and C, only four produce desired current pulsesupon a hybridization event.

As mentioned above, only a hybridization event produces a measurable(nanoseconds) current pulse at the nozzle. For optimized operation, thefollowing principles apply.

All excitation sources, detectors and stepped motion are synchronized.

Synchronized steps should be a fraction of the nozzle opening size(e.g., on the order of 5 nanometers).

Nozzle locations should be known with nanometer or sub-nanometerprecision in relation to a known reference position.

Nanometer alignment is very important to optimal operation.

Vibrations and other agitations should be minimized.

A sub-system is provided to measure very low amplitude nanosecondpulses.

For continuous real time measurement of millions, or even hundreds ofmillions, of base pairs, a wide dynamic range sub-nanometer stepper ispreferred.

To calibrate the system, it is desirable to use known samples.

In a preferred embodiment, the probes in the form of electrodeconductors and/or other stimuli are applied in a gated manner. Thisreduces the signal to noise ratio thereby allowing for increasedsensitivity and ability to resolve the sequence of the specimen.

Detection of a hybridization event may be accomplished in certainembodiments by observing variations in resonant capacitance. Forexample, an AC bias is imposed through a probe and a grounded platform(or alternatively AC bias may be imposed through the platform and theprobes are sequentially grounded). The AC bias will alternately depleteand accumulate the specimen. The change in capacitance ΔC is recorded,for example, using a lock-in technique. The measured value AC may be thevalue across the entire C-V curve when larger AC voltages are used, ormeasured value ΔC may be the differential capacitance dC/dV when smallerAC bias voltage is used. The variation in the load across the specimenoccurs due to characteristics of the portion of the specimen to beresolved such as a monomer on a polymer strand, or due to creation of ahybridization event when the probe includes a hybrid pair counterpart.This load variation changes the resonant frequency of the system.

Electrical conductors as probes according to preferred embodiments ofthe present invention, formed as described above with respect to FIGS. 2and 3 above (e.g., in the configuration with a very fine tip compared tothe back end, or a “knife edge”) also serves to lower the resistance ofthe conductor.

Various embodiments of stimuli application are possible. 1) voltageonly; 2) voltage plus light (AND gate) (light is a noise reductionmeans); 3) synchronization with gating, pulsed voltage, light, andcurrent gate leads to substantial noise reduction; 3a) controlledstepping; 3b) apply voltage and light (AND gate)—light of differentwavelengths to enhance inelastic tunneling current; 3c) apply currentgate (measure with ammeter); 4) kT (thermal energy) may be reduced underlow temperature operating conditions, e.g., T between 4 and 100K.

Gated detection serves to minimize noise and allow for preciseresolution of the extended object. Gated detection is necessary toensure the detection of picoamp level currents in the presence of noise.One effective strategy is to apply all of the stimuli in the propersequence, in the form of pulses. The pulse widths and heights areadjusted to achieve optimum results. The levels of voltage will be inthe 10 s of millivolts up to about 1 volt. The pulse durations may beabout 1 nanosecond to about 1000 nanoseconds, or longer if necessary.

The protocol for gated detection is described in the following steps: 1)apply a pulse to step the specimen relative to the platform to aposition to measure a portion of or a nucleotide of the specimen; 2)subsequent application of an electric field to provide contact betweenthe specimen and the probe; 3) optional application of a laser pulse; 4)application of tunneling device voltage pulse; 5) applying a pulse toopen the switch to the current measure device; 6) repeating 1-5 tomeasure the subsequent portion of the specimen or nucleotide tosequence. These steps 1-5 are synchronized pulses synchronized to amaster clock. In the event that particle beams are applied, orintensifiers, these will also have appropriately applied excitationpulses to activate them synchronized with said clock. These gatedsynchronized methods allow one to measure the detectable interactionwith a high signal to noise ratio.

For example, referring now to FIG. 77, a sampling period 7700 of aseries of synchronous excitations are charted on a plot 7702 relative toclock signals 7710. A stepping period is shown as a short pulsecommencing at a certain time indicated on a horizontal axis 7720, e.g.,at the start of the sequence. A contact period is shown as commencingafter the stepping period as indicated on a horizontal axis 7730 andending during of after measurement and/or processing and storageperiods. A photon period is shown as increasing in amplitude after thecommencement of the contact period as indicated on a horizontal axis7740 and ending proximate the end of the contact period. A voltage biasperiod is shown as commencing during the photon period as indicated on ahorizontal axis 7750 and ending proximate the end of the contact period.A current detection period is shown as commencing during the photonperiod and the voltage bias period as indicated on a horizontal axis7760 and ending proximate the end of the contact period. A processingand storage period is shown as commencing near the end of the photon,voltage bias and current detection periods as indicated on a horizontalaxis 7770 and ending after the end of the contact period.

Detection of the portion of the specimen under examination may occur byvarious contribution. In general, the detection schemes allow formolecular level (or detection of one or more monomers, or certain groupsof monomers, in an extended object to be analyzed) identification ofmonomers within a chain.

In a single strand specimen analysis systems having probes that induce ahybridization event, detection contribution includes elastic tunneling,inelastic tunneling, resonantly enhanced tunneling, and/or capacitance.FIG. 80 shows the typical Watson and Crick base pairing model. Referringnow to FIG. 81, a system 8105 schematically shown including a probe 8110and a substrate 20 having a specimen 8130 thereon. The probe is designedto induce a hybridization event, as described herein, by including acomplementary specimen in a well, on a substrate, or by otherconfigurations. A voltage bias is applied, for example, that correspondsto the N—H bonds and O—H bonds formed during a hybridization event.

The elastic tunneling contribution in systems having probes that inducea hybridization event is generally due to the tunneling interactionvariations that occur due to the distance between hybridized species.When a hybridization event occurs, the distance between the hybridizedmonomers (nucleotides) is modulated as the bond is created. As thetunneling barrier thickness decreases, tunneling probability increasesand thereby increases the tunneling contribution. This will bemanifested in the increase of conductance as measured in thecurrent-voltage characteristics of the hybrid bond. When nohybridization event occurs, the distance between the probe capable ofinducing a hybridization event and the specimen nucleotide remainsrelatively large, and hence the elastic tunneling contribution isrelatively low.

Referring now to FIG. 82, system 8205 schematically shown to illustratethe elastic tunneling contribution. When a bond is established as aresult of the relatively shorter distance (thinner tunneling barrier)that results for the H-Bond. This manifests itself as an increase of theconductance, and hence higher current. Note this elastic tunnelingcontribution generally does not involve exciting a resonance.

The inelastic tunneling contribution in systems having probes thatinduce a hybridization event is based on increased bond energies,especially hydrogen bond energies. During a hybridization event, aselectrons tunnel, the electrons lose energy by exciting the hydrogenbond created as a result of the hybridization event. This leads to atunneling contribution at a voltage correlating to the energy of thebond. When no hybridization event occurs, there is no hydrogen bondcreated, therefore there is no inelastic tunneling to excite such abond, and therefore no conductance contribution should be observed.

Referring now to FIG. 83, system ___05 schematically shown to illustratethe inelastic tunneling contribution. In addition to the above increasein current due to the elastic tunneling contribution, another increasewill be detected due to an inelastic tunneling contribution resultingfrom exciting the resonance of the H-Bond.

The above may enhanced by applying a source tuned to the bond frequency,thus providing an optically enhanced inelastic tunneling contribution.For example, as described above with regard to FIG. 28, a tune lightsource may be applied in conjunction with the measurement bias. Thisoptically enhanced inelastic tunneling component contributes tominimizing the noise effect by acting as and “AND” gate, such thatcurrent signal detection is primarily when synchronous application ofthe optical signal “AND” the bias voltage (both tuned to the resonance).

Referring now to FIG. 79, another embodiment of the present invention isshown. A specimen portion 7910 is within a probe system 7920 including afirst probe 7930 and a light nozzle 7950. The light nozzle 7950 and thefirst probe 7930 are activated, either sequentially, simultaneously oroverlapping in time, to facilitate current detection, measurement, orother impact of the detection contribution effect, as described above.The first prove 7930 may include any one of the above referenced typesof probes. Alternatively, more than one probe may be used with a coolingdroplet supply nozzle 2740, for example, for photonic application,current measurement, voltage bias, or other functionality as describedherein.

The resonantly enhanced tunneling contribution in systems having probesthat induce a hybridization event is based on measurement of excitedbond energies, particularly hydrogen bonds. Stimuli such as lightapplication is applied. A resonantly enhanced tunneling contribution maybe observed when a light source such as a laser having a suitably tunedwavelength excites the hydrogen bond created upon hybridization.Hydrogen bonds from the hybridization events can be excited by tuning alaser beam to the same energy as the bond. This will enhance thedetection of both the elastic and inelastic tunneling contribution andadd a resonant enhanced tunneling contribution to the measurementcurrent. Further, noise is minimized with suitable gating as describedherein since the pulsed application of the laser light source issynchronized with application of a voltage and during the opening of themeasurement current sensor. These simultaneous interactions have theeffect of a logical “AND” gate.

The capacitance contribution in systems having probes that induce ahybridization event is based on enhanced permittivity. Since thetunneling area is very small, the application of a laser beam tuned ator near the bond energy creates a resonantly enhanced permittivity atthe hybridized pair. This in effect is like a quantum capacitance. Thisquantum capacitance, added to a specific inductive element, an RFresonant circuit, or a RF resonant cavity, results when thehybridization even occurs. For example, the inductive element, RFresonant circuit or RF cavity are excited and can give a very largesignal. Since RF frequencies are at higher frequencies than the DCvoltages, there is low noise in that region (avoiding the 1/F noise).

Referring now to FIG. 84. system ___06 schematically shown to illustratethe quantum capacitance contribution. The quantum capacitancecontribution is a result of enhancement of polarizability of moleculesby exciting suitable resonances, including O-H or N—H bonds, and furtherrotational, vibration, and electronic. These are represented in Figure___ by resonances ω1, ω2, and ω3. The energy is represented by:

Eqc=½(Cq V2).

RF measurement is conducted using special resonance circuits thatinclude “quantum capacitance” which will be enhanced when O-H or N-Hresonances are excited by external radiation tuned to these resonances.This is expected because the capacitance is related to the permittivityof the interaction between the probe ___10 and the sample ___30. Thispermittivity has a susceptibility component which in turn is given bythe polarizability at the molecular level. The value of thispolarizability has many resonant contributions, including vibrational,rotational, and electronic. It is well known that if any one of theseresonances—vibrational, rotational, or electronic—are excited, even awayfrom the specific bonds, a significant increase in the polarizability,and hence the capacitance, results. The optimum tank circuit, e.g., inmicrowave or millimeter wave, will be excited and detected. Since theseare high frequencies, we will be far away from the 1/f noise regime,thus the signal to noise ratio is large.

In a single strand specimen analysis systems having probes that do notinduce a hybridization event, detection contribution includes inelastictunneling, resonantly enhanced tunneling, and/or capacitance.

Detection based on the elastic tunneling contribution is notparticularly effective without a probe that induces a hybridizationevent. Since the distance between the probe (in a system that does notinduce a hybridization event) and the specimen nucleotide reamingrelatively large, the elastic tunneling contribution is relatively lowfor all nucleotides. Therefore, an elastic tunneling contribution is notsuitable for measurement detection system when using probes that do notinduce hybridization events.

However, detection of measurement current variances due on inelastictunneling contribution may be used. Since there is no hybridizationevent (e.g., the probes are formed of conductors or other style thatdoes not induce a hybridization event), we rely on the inherentresonance of each nucleotide to be analyzed.

Further, the resonantly enhanced tunneling contribution is suitable,wherein a light source (e.g., laser wavelength) is tuned to the inherentunique resonances of the nucleotides to be analyzed. The nucleotides tobe analyzed are be excited by tuning a laser beam to that uniqueresonance, which will enhance the detection of the inelastic tunnelingcontribution and other contributions to the current measurement.Further, noise is minimized with suitable gating as described hereinsince the pulsed application of the laser light source is synchronizedwith application of a voltage and during the opening of the measurementcurrent sensor. These simultaneous interactions have the effect of alogical “AND” gate.

The capacitance contribution in systems having probes that do not inducea hybridization event is also based on enhanced permittivity analysis.Since the tunneling area is very small, the application of a laser beamtuned at or near the inherent unique resonance energies creates aresonantly enhanced permittivity of the signature. This in effect islike a quantum capacitance. This quantum capacitance, added to aspecific inductive element, an RF resonant circuit, or a RF resonantcavity, results when the signature energy occurs. For example, theinductive element, RF resonant circuit or RF cavity are excited and cangive a very large signal. Since RF frequencies are at higher frequenciesthan the DC voltages, there is low noise in that region (avoiding the1/F noise).

In other embodiments of the present invention, instead of, or inconjunction with, measuring a current variation,

Use probe, bring close to specimen, at known distance, attraction forcewill be detected. Rather than detect current flowing there through,detecting attractive or repulsive motion.

Knife edge AFM probe—contacts specimen, measures attractive or repulsiveforces

It is well known that atomic force microscopy (AFM) is used to analyzenano-structures an atomic scale. One key element leading to the successof the AFM is attachment of a nano-tip to a cantilever that is made todeflect when the nano-tip measures forces of the interaction betweensaid nano-tip an the structure under analysis. A laser beam reflectingfrom the cantilever measures the forces variations as the nano-tip scansthe structure.

By utilizing the inventive embodiments taught herein, it is possible toanalyze an extended object such as a DNA sequence by measuring the forceas in AFM, instead of or in conjunction with the tunneling currents.This is shown in Figure {AFM1}. Here the attractive force that resultswhen A bonds with T and C bonds with G as a result of hybridization isrelied upon to detect certain species. The specificity of the sequencingis accomplished by utilizing a probe with characteristics that allow itto attract certain species, such as by attaching poly-A, poly-T, poly-C,and poly-G oligomers to nano-edge probes, for example, as describedherein. Each of the 4 nano-edge probes is attached to a differentcantilever. A detector measures the deflection of each differentcantilever which modulates the reflection of laser beams of a differentwavelengths in the response to the interactive forces between the edgeor tip nano-probe and the specimen to be analyzed.

The AFM sequencing processes and systems described herein may be furtherdescribed by the following. An extended object such as a single strandDNA (SSDNA) is stretched and immobilized on a substrate. A sub-Angstromresolution translation stage moves the specimen relative to the set ofedge-nano-probes.

The edge nano-probe with the poly-A attached to it will experience andattractive force when it is proximate to or lands on the specimen with aT base. This force will modulate the reflection of the laser beam ofwavelength λ_(A) by the cantilever. The modulated reflected beamannounces the presence of T at that location with the aid of a detectorand processing electronics.

The edge nano-probe with the poly-T attached to it will experience andattractive force when it lands on the specimen with a Abase. This forcewill modulate the reflection of the laser beam of wavelength λ_(T) bythe cantilever. The modulated reflected beam announces the presence of Aat that location with the aid of a detector and processing electronics.

The edge nano-probe with the poly-C attached to it will experience andattractive force when it lands on the specimen with a G base. This forcewill modulate the reflection of the laser beam of wavelength λ_(C) bythe cantilever. The modulated reflected beam announces the presence of Gat that location with the aid of a detector and processing electronics.

The edge nano-probe with the poly-G attached to it will experience andattractive force when it lands on the specimen with a C base. This forcewill modulate the reflection of the laser beam of wavelength λ_(G) bythe cantilever. The modulated reflected beam announces the presence of Cat that location with the aid of a detector and processing electronics.

The edge nano-probes with the poly-A, poly-T, poly-C, or poly-G willexperience a weaker (no force or repulsive) force when either noncomplementary base, e.g. A on A, T on T, C on C, G, on G, A on C, A onG, T on C, or T on G. In these cases the beams reflected from thecantilevers will have small force modulation.

It is possible to use a single laser beam that is divided into 4beam-lets, each is focused on different cantilever at certain positions,to minimize interference. This detector will specially resolve thepositions of the beam-lets so as to differentiate and ensurespecificity.

Auxiliary laser beams may optionally be focused on the specimens, forexample, that are tuned to certain frequencies that interact with thespecimen. This can enhance the specificity and reduce errors ofambiguity.

Instead of using 4 nano-probes in parallel whereby each reflects its ownlaser beam or beam-let, it is possible to have nano-probes that areinserted or activated sequentially. For example, an embodiment is thissystem is illustrated in Figure {AFM2}. Here the probes are attached toa rotating mechanism (e.g., “daisy wheel style”) which rotates to exposethe probe to the specimen one at a time. To sequence a DNA specimen, theprobe functionalized with the poly-A oligomers is inserted (rotated in)and will scan the specimen. Then the poly-T is inserted to record thepositions of the A nucleotide. This is repeated for the C and Gnucleotides until the entire specimen is scanned with the four probesand the sequencing is completed. As shown in Figure {AFM3}, thisapparatus may be made more general and versatile by attaching to thedaisy wheel a plurality of probes with different shapes, knife edge,single point, multiple tips, different functional group to recognizespecific species, and nano-crystals of specific composition designed tosearch for and locate a specify material. This versatility isparticularly useful as it affords the opportunity to use the system asan imaging tools first, as in normal AFM, then as a sequencing tool ormore generally a chemical analysis tool.

It is appreciated that instead of a daisy wheel arrangement, there mayother more advantageous arrangements. In order for these apparatuseswith sequential insertion of probes to function properly, precisealignment subsystem may be required located with precision a spatialreference point, relative to which all spatial information is recorded.This will minimize errors and ambiguity. Additional nano-probes may beattached to function as the locators of alignment marks purposelywritten on the substrate.

As descried herein, array of probes sets in 2d or 3d arrays can measureand re-measure the same sample. This is possible due to the low costtechniques. Further, multiple channels for parallel systems may be used.

As descried herein, array of probes sets in 2d or 3d arrays can measureand re-measure the same sample. This is possible due to the low costtechniques. Further, multiple channels for parallel systems may be used.

In another embodiment, and referring now to Figure {DD1}, a system isprovided to use differential detection to minimize errors in reading thesequence. Arrays of nano probes/nozzles affords the opportunity,inexpensively, to consider repeated measurements to minimize the noise.For example, differential detection strategies may be used wherebysystem noise may be subtracted in real time. One or more probes or probesets read the specimen and known samples A,C,T,G. Accuracy may beincrease by performing differential detection, whereby noise may bedetermined and subtracted from the specimen reading. For example, we mayread synonyms with the specimen analysis a current of a known sample(e.g., Arrays of A, C, T, and G). This gives us noise and thecontribution of T at a particular instant of time. At the same instantof time, if a T is apparently determined to be the base of the specimen,the noise may easily be subtracted to confirm that the reading of T isaccurate. Therefore, the following apply:

Current(known sample)=noise+contribution of T(apply positive pulse)

Current(specimen)=noise−contribution of T(apply negative pulse)

The contribution of the signal is detected at certain modulationfrequency, whereas the noise is random

AAA, GGG, TTT, CCC also could be known AGAGAGAG, TCTCTCTC, so long as itis known.

Many sensing techniques for determining a hybridization event includeelastic quantum mechanical tunneling; inelastic quantum mechanicaltunneling; resonantly enhanced tunneling; resonantly enhanced quantumcapacitance in a tank circuit to boost the signal of hybridizationevents; fast cooling techniques to reduce noise (for example, such asthe system that utilized liquid He or liquid N₂ droplet cooling); ionicconductivity; quantum mechanical tunneling electron emission; photonemission, which can be amplified by photon multiplier techniques. Anyone or more of these techniques may be used in conjunction with theherein described high spatial resolution (e.g., nucleotide monomer levelresolution) probes, probe sets or probe arrays as a novel directsequencing system.

In certain embodiments, fast cooling techniques may be incorporated. Asshown in FIG. 78, for example, a specimen portion 7810 is within a probesystem 7820 including a first probe 7830 and a cooling droplet supplynozzle 7840. The cooling droplet supply nozzle 7840 may include liquidHe, liquid N2, or other suitable coolant suitable for fast coolingapplication. The first probe may include any one of the above referencedtypes of probes. Alternatively, more than one probe may be used with acooling droplet supply nozzle 7840, for example, for photonicapplication, current measurement, voltage bias, or other functionalityas described herein.

Another aspect of the present invention to minimize error is theextended configuration (e.g., “knife edge”) as described above withrespect to FIGS. 2 and 3.

In systems herein where metal contacts or probes are used to measurecurrents and voltages from small structures such as the monomers of thespecimen, four probe tunneling devices may be used (e.g., shown in FIG.17) to minimize contact and lead resistance. Also, preferred probeconfiguration provide for a larger end opposite the tip, for example, asshown with respect to FIG. 2. Further, all contacts the probe arepreferably much larger than the tip. This can, for example, reduceelectrical resistance of the probe when end serves as a contact region.

Optimum specimen resolution and speed may be achieved by optimizing thedetection system to increase the measurable signal, namely, ensuringthat enough electrons are involved, and minimizing the ambient noise.The tunneling current densities involved, in such small tunneling areas(e.g., 0.5 square nanometers), makes it possible to involve 10 s ofelectrons and 10 s of picoamps. This is achieved by allowing the timeaperture to excite and detect each nucleotide in the order of 1-1000nano-seconds. This can achieve the desired result of sequencing thewhole Human Genome of 3×10⁹ base pair in a time of about 1 second to afew minutes.

We have allowed for even higher speed and fewer electrons to be involvedwhereby intensification/amplification sub-systems are used to intensifyfew electrons or photons into a measurable signal.

Gated electronic techniques are also used herein with a pulse protocolthat is applied to ensure minimize noise. This is desirable to ensurethe detection of picoamp level currents in the presence of noise. Oneeffective strategy is to apply all of the stimuli in the propersequence, in the form of pulses. The pulse widths and heights areadjusted to achieve optimum results. The levels of voltage will be inthe 10 s of millivolts up to about 1 volt. The pulse durations may beabout 1 nanosecond to about 1000 nanoseconds, or longer if necessary.

The protocol for gated detection to minimize noise is described in thefollowing steps: 1) apply a pulse to step the specimen relative to theplatform to a position to measure a portion of or a nucleotide of thespecimen; 2) subsequent application of an electric field to providecontact between the specimen and the probe; 3) optional application of alaser pulse; 4) application of tunneling device voltage pulse; 5)applying a pulse to open the switch to the current measure device; 6)repeating 1-5 to measure the subsequent portion of the specimen ornucleotide to sequence. These steps 1-5 are synchronized pulsessynchronized to a master clock. In the event that particle beams areapplied, or intensifiers, these will also have appropriately appliedexcitation pulses to activate them synchronized with said clock. Thesegated synchronized methods allow one to measure the detectableinteraction with a high signal to noise ratio.

In another embodiment, referring now to FIG. 8C, a plurality ofnano-probe sets are provided, wherein each nano-probe set is specific toa certain species (e.g., nucleotide). The specimen is measure severaltimes (by each probe within the probe set) and stored by a the firstsingle species probe set. The specimen is then sequentially measuredwith a second single species probe set, a third single species probeset, and a fourth single species probe set to obtain data from eachgroup of probe sets and obtaining at least one hybridization event orother detection event, preferably duplicate events to ensure accuracy ofdetermination. Each probe set may and the computer analysis provides aconsensus of the identity of the species, after averaging or othersuitable statistical analysis. Each species is measured several times byone group, then another, then the 3rd, then the 4th. For example, if aprobe set is optimized to detect an event with a T species, thefollowing detection readout may be determined at that probe set for thatbase: TTCT. As the specimen and hence a particular base is moved acrossthe array of 4 probe sets, A/T/C/G, the following detection readout maybe determined at that probe array for that base: TTCT/- -G -/C- - -/--A-. Thus, some of the individual probes within the sets may provideerroneous results (e.g., the C within the first TTCT, the G within thesecond group, the C within the third group and the A within the fourthgroup), statistical analysis will determine that the particular base isindeed a T base. Note that more or less probes that four may be in eachprobe set. Further a scheme may be provided with various degrees ofredundancy, including differing numbers of probes within the probe sets,combinations of homogeneous and heterogeneous probe sets, combinationsof probe type for various detectable Interactions (e.g., nucleotidefilled wells, solid state nucleotides, metal conductor, metal plus knownnucleotide stand, open well or funnel for particle beams, electron beamemission, ion beams, x-rays or the like, or flexible membrane probes.

One important factor of these method and strategies for error reductionis obtaining a sufficient signal to noise ratio. The system ispreferably gated and synchronized such that the ammeter will only detecta signal when a nucleotide is directly below a nozzle. The bias appliedmay be positive, negative, or even alternating, as to maximize thechange in conductivity. Cooling may be desirable to reduce the thermalnoise. Alternatively, each DNA or protein strand may be passed underseveral arrays of nozzles, thereby averaging out the noise. Certainembodiments show array configurations, e.g., that may average out noiseand increase SNR. These features will help in assuring an excellent SNR.

However, if we assume a 10 picoamp current change under one appliedvolt, and 10 nanoseconds for detection, the signal is orders ofmagnitude larger than the thermal noise, even at room temperature. Thesequencing speed would be enormous. Allowing 30 nanoseconds to move anozzle from one nucleotide to the next (a speed of about 1 cm/sec), itwould take only 40 nanoseconds to sequence one base pair, which isequivalent to 1.5 Billion base pairs a minute.

In certain embodiments, fast cooling techniques may be incorporated. Asshown in FIG. 27, for example, a specimen portion 2710 is within a probesystem 2720 including a first probe 2730 and a cooling droplet supplynozzle 2740. The cooling droplet supply nozzle 2740 may include liquidHe, liquid N2, or other suitable coolant suitable for fast coolingapplication. The first probe may include any one of the above referencedtypes of probes. Alternatively, more than one probe may be used with acooling droplet supply nozzle 2740, for example, for photonicapplication, current measurement, voltage bias, or other functionalityas described herein.

Referring now to FIG. 28, another embodiment of the present invention isshown. A specimen portion 2810 is within a probe system 2820 including afirst probe 2830 and a light nozzle 2850. The light nozzle 2850 and thefirst probe 2830 are activated, either sequentially, simultaneously oroverlapping in time, to facilitate current detection, measurement, orother impact of the detection contribution effect, as described above.The first probe 2830 may include any one of the above referenced typesof probes. The light nozzle 2850 may provide various types photonicenergy, for example, visible, UV, X-Ray, THZ, IR, or FRIR.

In other embodiments described herein, and referring back to FIGS.6A-6F, the probes may be oriented at various angles with respect to thespecimen. Referring to FIGS. 6A and 6B, all probes and probe setsdescribed herein may be configured with respect to the specimen atvarious angles. For example, referring to FIG. 6A, a probe set 630 maybe oriented generally perpendicular (in the length direction) to aspecimen 650. Further, referring to FIG. 6B, a probe set 630 may beoriented (in the length direction) generally at an angle θ with respectto a specimen 650. Referring to FIG. 6C, a system 660 is presentedwhereby the orientation of plural probe sets 630 relative a specimen 650varies. Because the objects of the specimen 650 (e.g., bases within aDNA strand) may have different orientations, it may be desirable tosequence with a plurality of probe sets 630. The plurality of probe sets630 may have different angles θ₁, θ₂, θ₃, θ₄, θ₅, . . . θ_(n) (e.g., 20°to 160° in suitable increments, arranged sequentially, randomly or inanother desirable arrangement. During measurement as described furtherherein, a controller may determine which orientation of the probe setyields the best signal for a particular base at its inherentorientation. This allows one to measure the data from the probe sets ofthe array, and determine the optimum signal for certain bases or groupsof bases. In another embodiment, and referring to FIGS. 6D-6F, theangles of orientation in the height direction may also be varied. Forexample, referring to FIG. 6D, probe set 630 may be oriented in theheight direction generally perpendicular (90° with respect to thespecimen 650. Further, as shown in FIG. 6E, probe set 630 may beoriented in the height direction generally at an angle ω with respect tothe specimen 650. Referring to FIG. 6F, a system 670 is presentedwhereby the orientation in a height direction of plural probe sets 630relative a specimen 650 varies. Because the objects of the specimen 650(e.g., bases within a DNA strand) may have different orientations, itmay be desirable to sequence with a plurality of probe sets 630. Theplurality of probe sets 630 may have different angles ω₁, ω₂, ω₃ . . .ω_(n) (e.g., 20° to 160° in suitable increments, arranged sequentially,randomly or in another desirable arrangement. By measuring at thesevarious angles, the opportunities for errors and misreading areminimized or eliminated.

In another embodiment, and referring now to Figures {PFM}A and {PFM}B, abendable membrane material {PFM} 10 having a nano-scale probe attachedthereto is provided. The nano-scale probe {PFM} 12 may one of theaforementioned probes such as a known nucleotide strand, functionalizedgroup, or other molecular probe. Preferably the bendable membranematerial {PFM} 10 include a metallic surface with the probe {PFM} 12attached thereto to facilitate current measurement. Using a suitableMEMS device or other plunger {PFM}20, a flexible metal membrane {PFM} 16is pulsed to make contact with the specimen {PFM}40 to resolve it.

As with the other probe types described herein, a 2D or 3D array may beprovided. Further, these arrays may include homogeneous or heterogeneousprobe types.

Furthermore, in general, the probe may make contact with the assistanceof other known devices such as angstrom or sub-angstrom precisionactuators, MEMs devices, or other mechanical devices.

Referring now to Figure {TS1}, a structure {TS1} 05 is shown thatfacilitates attraction and transport polymeric structures such as DNAfragments, RNA molecules, proteins, or other polymeric structure. Asubstrate {TS1} 10 is provided with one or more coaxing lines {TS1}20.These coaxing lines or regions may be in the form of channels, channelsincluding a suitable coaxing material, lines or regions of the surfaceof the substrate {TS1}10 treated with a suitable coaxing material, aridge or other protrusion defining the one or more coaxing lines{TS1}20, or a ridge or other protrusion defining the one or more coaxinglines {TS1}20 treated with a suitable coaxing material. A coaxingmaterial may include materials such as amino-silane, biotin, other knownbonding materials, charged conductive particles such as platinum, goldor other suitable material.

In general, a the specimens may include magnetic portions, or suitablechromophores or fluorophores to help guide and manipulate the specimens.

Note that the substrate {TS1}10 may be in the form of a glass slide,e.g., on the order of 1-2 cm by 3-5 cm. Alternatively, the substrate{TS1}10 may be in the form of a disc or wafer. The form factor of theslide will generally be a function of the analysis tools and/ormanipulation tools used to work with the specimen.

This structure {TS1}05 may be used with DNA sequencing tools, forexample, described in conjunction with U.S. patent application Ser. No.10/775,999 filed on Feb. 10, 2004 entitled “Micro-Nozzle, Nano Nozzleand Manufacturing Methods Therefor”, U.S. Provisional Patent ApplicationSer. No. 60/669,029 filed on Apr. 7, 2005 entitled “DNA SequencingMethod and System”, and U.S. Provisional Patent Application Ser. No.60/699,619 filed on Jul. 15, 2004 entitled “Molecular Analysis Probe,Systems and Methods, including DNA Sequencing”, all of which areincorporated by reference herein.

Further, these structures {TS1}05 may be used with various other typesof analytical tools such as optical imaging tools. Certain usefuloptical imaging tools that may benefit from the structures {TS1} 05described herein are described in U.S. patent application Ser. No.10/800,148 filed on Mar. 12, 2004 entitled “Microchannel Plates AndBiochip Arrays, And Methods Of Making Same” and U.S. Provisional PatentApplication Ser. No. 60/674,012 filed on Apr. 22, 2005 entitled“Microchannel Plate And Method Of Making Microchannel Plate”, all ofwhich are incorporated by reference herein.

Referring now to Figure {TS2}, a structure {T52}05 is shown thatfacilitates attraction and transport polymeric structures such as DNAfragments, RNA molecules, proteins, or other polymeric structure. Asubstrate {TS2} 10 is provided with a plurality of coaxing lines{TS2}20.

Referring now to Figure {TS3}, a structure {TS3}05 is shown thatfacilitates attraction and transport polymeric structures such as DNAfragments, RNA molecules, proteins, or other polymeric structure. Asubstrate {TS3}10 is provided with one or more virtual coaxing lines{TS3}25 defined by plural electrodes {TS3}30 therealong. These virtualcoaxing lines or regions may be in the form of channels with suitableelectrodes {TS3}30, virtual lines or regions on the surface of thesubstrate {TS3}10 with suitable electrodes {TS3}30, a ridge or otherprotrusion defining the one or more virtual coaxing lines {TS3}20 withsuitable electrodes {TS3}30. Accordingly, with plural discontinuouselectrodes {TS3}03, the virtual coaxing line {TS3}25 is defined. Theelectrodes in these embodiments may include pre-charged particles,include an on-board battery, or include electrodes that are activated bysuitable devices with the system reader.

Referring now to Figure {TS4}, a structure {TS4}05 is shown thatfacilitates attraction and transport polymeric structures such as DNAfragments, RNA molecules, proteins, or other polymeric structure. Asubstrate {TS4} 10 is provided with one or more coaxing lines {TS4}20having plural electrodes {TS4}30 therealong. These coaxing lines orregions may be in the form of channels, channels including a suitablecoaxing material, lines or regions of the surface of the substrate{TS4}10 treated with a suitable coaxing material, a ridge or otherprotrusion defining the one or more coaxing lines {TS4}20, or a ridge orother protrusion defining the one or more coaxing lines {TS4}20 treatedwith a suitable coaxing material, wherein the coating material may bethe same as those described above, or alternatively may includematerials that have attraction forces when subjected to the electricfields created by the electrodes {TS4}30.

In certain embodiments, an electric field may be applied at a desiredstart position {TS4}40 on the structure {TS4}05. Further, in the variousembodiments of the structures that facilitate attraction and transportof specimens, various features may be aligned to other system featuresdescribed herein.

For example, and referring now to Figures {TS5}A-{TS5}G, a method ofcoaxing strands onto a structure {TS1}05, {TS2}05, {TS3}05 or {TS4}05. Astructure {TS5}05 is inserted into a solution containing one or morepolymeric structures such as DNA strands or fragments. One or morefragments will attach to said structure {TS5}05 as shown by arrows inFigure {TS5}C. Referring to Figures {TS5}D-F, structure {TS05}05 havingone or more polymeric strands attached thereto is then pulled out of theliquid. Preferably, the structure {TS05}05 is removed in a directionalong the axis of the coaxing line such that the liquid flow directionand gravity also contribute to the attractive forces of the coaxinglines. Accordingly, since the liquid flow forces, gravitational forcesand the contribution of the coaxing line are in substantially the samedirection, the strands are coaxed toward alignment. In certainembodiments, an electric field may be applied at a desired startposition on the structure {TS05}05.

To assist the denaturing in conjunction with the precise stepwisemotion, the DNA strand can be straightened bay various methods. In oneembodiment, electrostatic fields may be used to attract the negativelycharged strands. In another embodiment, a magnetically attractive beadmay be applied to an end of the DNA strand, and the strand pulled withmagnetic force. In a further embodiment, viscosity optimization may beemployed, such that while dragging the strand through a liquid proximateor in the channel, it will straighten upon optimal dragging velocity andfluid viscosity conditions. Further, hydrophilicity may be used, e.g.,by suitable material treatment at or in the nozzles and channel walls,to attract nucleotides. In other embodiment, hydrophobicity may be used,e.g., by suitable material at or in the nozzles and channel walls, tomaintain the fluid within the channel.

Referring now to Figure {CS1}, an overview of a coarse shuttle system{CS1}10 is shown. System {CS1} 10 serves to facilitate displacement ofthe extended object {CS1}20, and in particular to move and stretch anextended object {CS1}20 such as a DNA or RNA strand or fragment througha path {CS1}14 (which may be a channel or along the surface of asubstrate) between two sides {CS1}30, {CS1}40.

In general, each side {CS1}30, {CS1}40 has a plurality of electrodepairs arranged about the path {CS1}14. For example, as shown in Figure{CS1}, the channel 14 includes a wider opening area {CS1} 16, forexample, to increase the likelihood of extended object {CS1}20encountering the channel {CS1}14. Electrode pairs {C51}31, {CS1}41through {C51}38, {CS1}48 are arranged on the sides {CS1}30, {CS1}40. Inthe example where the extended object {CS1}20 is a negatively chargedextended object, such as a DNA strand, positive charges are appliedacross the Electrode pairs {CS1}31, {CS1}41 through {CS1}38, {CS1}48,thereby coaxing the extended object {CS1}20 into and through the path{CS1}14.

Note that the path {CS1}14 may be in the form of a channel, e.g., havingpartially enclosed walls such as a concave groove, V-shaped groove,U-shaped groove, or other suitable shape. Alternatively, the path{CS1}14 may instead be defined by suitable surface treatment, asdescribed further herein. Alternatively, .the path {CS1}14 may be anelevated ridge treated or pattered with electrodes, either along thesides as shown with respect to the molecular shuttle herein or along allor portions of the length of the path {CS1}14.

Figures {NS1}A-C show an embodiment of a molecular shuttle {NS1}07, forexample, for fine displacement of an extended object {NS1} 12. Ingeneral, the molecular shuttle {NS1} 07 may be used to controllablydisplace an extended object {NS1} 12, for example, from a first location{NS1} 16 to a second location {NS1} 18 to a third location {NS1}20, andso on. The extended object {NS1} 12, such as a DNA strand, DNA fragment,RNA molecule, protein molecule, or various other types of polymer andextended object, is typically charged, in this case shown as negativelycharged. The molecular shuttle {NS1} 07 includes a plurality ofspatially opposing probes {NS1}22, {NS1}24 within or upon substrates orsubstrate regions 26, 28 thereby defining a path {NS1}30 therebetween.In certain preferred embodiments, these probes {NS1}22, {NS1}24 areformed as probes as described herein. As shown in Figure {NS1}A, theextended object {NS1}12 is outside of the path {NS1}30. By applying apositive charge at probes {NS1}22, {NS1}24 at the end of the molecularshuttle {NS1}07 (as indicated by “+” signs in Figure {NS1}A), theextended object {NS1} 12 will be attracted to an opening {NS1}32 of thepath {NS1}30.

Referring to Figure {NS1}B, when another positive charge is appliedthrough the probes {NS1}22, {NS1}24 at a location indicated by line{NS1} 18, with negative charges provided by probes or electrodes betweenposition {NS1} 18 and the positive charge at opening {NS1} 32, theextended object {NS1} 12 will be attracted to the position {NS1} 18within the channel path {NS1} 30. Referring to Figure {NS1} C, theprocess is continued to shuttle the extended object {NS1} 12, forexample, to a position {NS1}20 within the path {NS1}30.

Referring now to Figures {NS2}A-{NS2}D, a molecular shuttle {NS2}07 maybe formed of various shapes, including but not limited to a curved orsemicircle channel (Figure {NS2}A), a Y-shaped channel (Figure {NS2}B),a series of channels directed in a radial manner to or from a centralpoint (Figure {NS2}C), or T-shaped (Figure {NS2}D), for example.

Note that the path {NS1}30 may be in the form of a channel, e.g., havingpartially enclosed walls such as a concave groove, V-shaped groove,U-shaped groove, or other suitable shape. Alternatively, the path{NS1}30 may instead be defined by suitable surface treatment, asdescribed further herein. Alternatively, .the path {NS1}30 may be anelevated ridge treated or pattered with electrodes, either along thesides as shown with respect to the molecular shuttle herein or along allor portions of the length of the path {NS1}30.

Referring now to FIG. 26, a reference position and precision nanometermetrology system is shown. A reference position probe (RPP), e.g.,formed of platinum or other suitable material, or in the form of anano-light guide, or other excitation probe structure, is included inthe probe set or nanonozzle array set. The positions of each probe ornanonozzle relative the RPP is known. This reference position probeprovides a known starting point when sequencing commences for precisemetrology.

Referring now to FIG. 75, the stepped motion of ssDNA is shown relativeto a known position of the RPP.

In certain embodiments, the specimen may be within a channel of thebase. A channel may include suitable fluid, or the specimen may becoaxed through a channel with little or no fluid.

In other embodiments, the specimens may be embedded within the base,e.g., in a biochip.

In certain embodiments, an electron or photon intensifier such as amicro-channel intensifier may be used. For example, referring to FIGS.16A and 16B, these embodiments are shown.

Referring to FIG. 16A, the probe emitter interacts selectively with thespecimen in an elastic or inelastic manner, whereby energy is lost, andthe event lead to the release of photons or electrons that have specificenergy indicative of the nature of the molecule or monomer. Theseelectrons or photons may be too few to be measured directly. Therefore,the invention herein provides for an intensification or amplificationsub-system such as micro-channel plate intensifiers known in the art,e.g., night vision goggles or photo-multipliers.

Referring now to FIG. 16B, where the probe is either metallic and/or amolecular probe, interaction with the specimen may be through inelastictunneling current. Rather than measuring this tunneling currentdirectly, it is possible to provide a sub-system for allowing eitherphotons or electrons to be emitted. The photons or electrons to beemitted may occur upon a hybridization event, or by applying suitablevoltage energy to emit inelastic electrons indicative of the spectra ofthe specimen. This electron is also detected my anintensifier/amplification sub-system described above with respect toFIG. 16A.

Referring now to FIG. 16C, an array of intensifiers/amplifiersub-systems as described with respect to FIG. 16A or 16B may beprovided. For example, the exciting probe beams or other probe types maybe tuned or optimized from a particular monomer, for example, in a DNAsequencing system, A, T, C, G, such that the electrons or photons areemitted are signatures of each type of nucleotide to be detected.

Sequencing extended objects including but not limited to DNA, RNA,proteins in general, other polymers, oligomers, and other nano-scalestructures. Thus, as shown and described, the herein system includingnano-nozzles and nano-nozzle arrays are very well suited for ultra fastreal time DNA sequencing operations.

In addition to sequencing or analyzing DNA strands or fragments, probesand systems according to the present invention may be used for varioustypes of extended objects including but not limited to DNA, RNA,proteins in general, other polymers, oligomers, and other nano-scalestructures.

Referring now to Figure {MAN1}, a probe {MAN1}02 having extremely smalltip dimensions t (or array or set of such probes) may be used as ageneral purpose manipulator for manipulating materials on the molecularor atomic level. For example, using the probe {MAN1}02 provides for ahigh field strength, in part due to its symmetry. This high field thatis advantageously localized due to the small probe dimensions willenable attraction of DNA strands, proteins, graphene layers,nanoparticles, other molecules, mono-molecular layers, or N such layers.

Referring to Figure {LITH1}, a general system is depicted for using theherein probes for ultra high resolution nanolithography. A probe set maybe provided, for example, wherein each probe includes the same ordifferent materials. In further embodiments, three-dimensionalnanostructures may be fabricated using the probes herein.

Figures {NS1}A-C show an embodiment of a molecular shuttle {NS1} 07. Ingeneral, the molecular shuttle {NS1} 07 may be used to controllablydisplace an extended object {NS1} 12, for example, from a first location{NS1} 16 to a second location {NS1} 18 to a third location {NS1}20, andso on. The extended object {NS1} 12, such as a DNA strand, DNA fragment,RNA molecule, protein molecule, or various other types of polymer andextended object, is typically charged, in this case shown as negativelycharged. The molecular shuttle {NS1} 07 includes a plurality ofspatially opposing probes {NS1}22, {NS1}24 within or upon substrates orsubstrate regions 26, 28 thereby defining a channel {NS1}30therebetween. In certain preferred embodiments, these probes {NS1}22,{NS1}24 are formed as probes as described herein. As shown in Figure{NS1}A, the extended object {NS1} 12 is outside of the channel 30. Byapplying a positive charge at probes {NS1}22, {NS1}24 at the end of themolecular shuttle {NS1}07 (as indicated by “+” signs in Figure {NS1}A),the extended object {NS1} 12 will be attracted to an opening {NS1} 32 ofthe channel.

Referring to Figure {NS1}B, when another positive charge is appliedthrough the probes {NS1}22, {NS1}24 at a location indicated by line{NS1} 18, with negative charges provided by probes or electrodes betweenposition {NS1} 18 and the positive charge at opening {NS1} 32, theextended object {NS1} 12 will be attracted to the position {NS1} 18within the channel. Referring to Figure {NS1} C, the process iscontinued to shuttle the extended object {NS1} 12, for example, to aposition {NS1}20 within the channel.

Referring now to Figures {NS2}A-{NS2}D, a molecular shuttle {NS2}07 maybe formed of various shapes, including but not limited to a curved orsemicircle channel (Figure {NS2}A), a Y-shaped channel (Figure {NS2}B),a series of channels directed in a radial manner to or from a centralpoint (Figure {NS2}C), or T-shaped (Figure {NS2}D), for example.

Referring to Figure {AS1}, a method is shown to use the probes accordingto the present invention to create atomically smooth surfaces. Forexample, a probe {AS1} 10 with an attached voltage source is swept overa surface {AS1}50. In the configuration of the probe as shown in Figure{AS1}, the probe produces a very high localized field strength. Thisfield can be used to sweep a surface to make it atomically smooth.

Another embodiment of the present invention exploits the ability to makeatomically smooth ultra-thin films as taught in the present inventionFigures {SLG 31B. These films can used as flexible substrates foranalyzing or sequencing unknown specimens. As shown in Figure {AFTM1},this flexible membrane may replace the flexible cantilevers in Figures{AFM1}-{AFM3}. Figure {AFTM1} shows a system {AFTM1}10 a membrane{AFTM1}12 between supports {AFTM1}14. As the specimen {AFTM1}30 passesunder a probe {AFTM1}20, atomic interactions occur, generally asdescribed above with respect to Figures {AFM1}-{AFM3}. However, theprobes {AFTM1}20 are fixed, thus the membrane {AFTM1} 12 is deflected bythose atomic forces. The deflection of the membrane {AFTM1} 12, inresponse to atomic forces, is detected by measuring the reflection ofthe incident laser beam {AFTM1}40 on the membrane {AFTM1} 12. Byseparating the deflection from the probe, a more general purposeapparatus results, namely, combing AFM capability with STM imaging aswell sequencing tools all in one device. As shown in {AFTM1}, one ormore probes {AFTM1}20 are connected to suitable voltage sources and thesupports {AFTM}14. Other stimuli may also be provided for certainapplications, such as scanning tunneling and other sequencingfunctionality. For specificity, the prove may be a specifically formedprobe, such as a nucleotide specific probe as described above. A deviceparticularly suited for sequencing DNA strands includes one thatincorporates at least a set of 4 probes, include nucleotide specificprobes for A,C,T and G, for example, in a configuration as describedherein with respect to Figure {AFM2}, with the flexible membrane {AFTM1}12.

This membrane deflecting apparatus allows for the possibility ofreplacing the laser beam with a parallel conducting plate directlyunderneath the membrane separated by an appropriate distance. As shownin Figures {AFTM2}A-{AFTM2}B, this forms a capacitance that variesaccording to the deflection of the membrane. Figures {AFTM2}A-{AFTM2}Bshow the deflection of the substrate membrane in response to the forcesat different probe positions. Therefore, the capacitance value variationor modulation can be related to the atomic forces experienced by themembrane. This happens because the fixture holding the probes is heldsubstantially fixed, thereby forcing only the membrane to respond to theforces.

The capacitance value is designed to be in the range of 0.1 to 10nano-Farad so that it can be part of a resonant circuit, Figure{AFTM2}C, comprising an inductance to oscillate at frequencies in theranges of 10 KHz-1 MHZ or 1 MHz to several GHz. By coupling an tunablesweep oscillator, it is possible to monitor the power absorbed by thesystem as a function of frequency.

Fig. {AFTM2}D shows the intensity, I_(ω) may be plotted as a function offrequency, ω=(LC)^(1/2), for different probe positions. Measuringfrequency shifts can be related to the capacitance variation thatresults from the varying forces F_(ω) at different positions. Figure{AFTM2}E illustrates the dependence of the F_(ω) on the frequency forattractive and repulsive forces. In a first position, the probeexperiences a repulsive force, causing the capacitance to decrease, andshifting the frequency to ω₁. In a second and third probe positions, theforces are attractive, shifting the requires upward to ω₂ and ω₃respectively.

Figure {AFTM3}A illustrates yet another embodiment of the presentinvention whereby a tool for analyzing specimens including specificapplication of sequencing DNA, RNA and atomic force imaging is provided.A probe according the teachings of the present invention is attached toa flexible membrane or cantilever. According to the exploded view inFig. {AFTM3}B, a first thin film inductor connected to a first thin filmplat of a capacitor are deposited on the flexible membrane on thesurface opposite the probe. A second thin film inductor connected to asecond thin film plate of a capacitor are deposited on a rigid member onthe surface facing the flexible membrane. The rigid member and flexiblemembrane and attached to each other with a suitable spacer having athickened that determines a desired capacitance value. The spacer alsomay contain an integrated circuit for processing and/or analyzing thesignals which result from the interaction of the probe with thespecimen. This signal is manifested in the variation of the capacitanceas a result of the forces that cause the membrane deflection. Figure{AFTM3}C shown the circuit model for analysis and processing. Similardetection principles apply as those employed in the apparatus describedFigures {AFTM2}A to {AFTM2}E.

This integrated atomic force probe can be used as in conventional ATMmodes, as well as for sequencing. The latter is accomplished bysequentially inserting different integrated probes functionalized tospecify different nucleotides. Alternatively, it is preferred tointegrate several capacitive probes in a single structure to performparallel sequencing and analysis functions as shown in Figure {AFTM4}.This fully integrated system allows the flexibility to have probes ofdifferent shapes and functionalized to recognize predetermined certainspecimens. The system can be addressed to select one of many modes,including but not limited to STM, AFM, sequencing, magnetic analysis, orother suitable functionalities, because it has a uniqueactivation/deactivation feature. This is accomplished with an integratedcircuit that supplies a DC voltage to the plates of the capacitor thatis selected to deactivate. This causes the flexible membrane to beattached permanently to the upper rigid plate. The removal of the DCvoltage releases the membrane and selects it and its probe foractivation.

Figure {AFTM5} illustrates the system of Figure {AFTM4} furtherincluding nucleotide specific probes for increases specificity, forexample, particularly suitable for imaging, analyzing and sequencing DNAspecimens.

The fully integrated probe illustrated in Figures {AFTM3}-{AFTM5} can beadvantageously manufactured by the methods and systems described inApplicant's multi-layered manufacturing methods, as described in U.S.Non-provisional application Ser. No. 09/950,909, filed Sep. 12, 2001entitled “Thin films and Production Methods Thereof”; 10/222,439, filedAug. 15, 2002 entitled “MEMs And Method Of Manufacturing MEMs”;10/017,186 filed Dec. 7, 2001 entitled “Device And Method For HandlingFragile Objects, And Manufacturing Method Thereof”; PCT ApplicationSerial No. PCT/US03/37304 filed Nov. 20, 2003 and entitled “ThreeDimensional Device Assembly and Production Methods Thereof'; U.S. Pat.No. 6,857,671 granted on Apr. 5, 2005 entitled “Method of FabricatingVertical Integrated Circuits”; U.S. Non-provisional application Ser.Nos. 10/717,220 filed on Nov. 19, 2003 entitled “Method of FabricatingMulti Layer MEMs and Microfluidic Devices”; 10/719,666 filed on Nov. 20,2003 entitled “Method and System for Increasing Yield of VerticallyIntegrated Devices”; 10/719,663 filed on Nov. 20, 2003 entitled “Methodof Fabricating Multi Layer Devices on Buried Oxide Layer Substrates”;all of which are incorporated by reference herein. However, other typesof semiconductor and/or thin film processing may be employed.

While the above examples apply to the sequencing of DNA, it isappreciated that the probes can be functionalized to have the ability torecognize other molecules with precise specificity making these methodsmore general for the recognition and analysis on unknown chemicals. Itwill have applications not only as a scientific tools, but also formedical as well as for sensing hazardous materials.

It is known that the replication and transcription of DNA involves theseparation of the two strands to reveal the base sequence of the singlestand to be replicated or transcribed. This is accomplished with ahelicase enzyme which causes the complementary strands to separate in afirst position to complete the transcription or replication processes.When this is completed, the two complementary strands bind again and thehelicase separates them at a second adjacent position to repeat theprocess. This is repeated along the entire DNA length until thereplication or transcription is done.

The present invention which teaching the analysis of an extended objectin general, and a single DNA strand sequence in particular, may beextended to also sequence double strand specimens. This may beaccomplished according the embodiments of the present invention bycausing the nano-probe to interact with the nucleotide bases in themajor and minor groves of the helical structure of the DNA strand orfragment. This process may optionally be facilitated further by the useof a suitable catalyst or enzyme such as helicase to cause localseparation of the complementary strands to reveal the bases to besequenced and to cause them to interact optimally with the nano-probe asdescribed herein. The catalyst or enzyme may be attached to or dispensedfrom the analyzing nano-probe or attached to or dispensed from anauxiliary nano-probe or nano-funnel in close proximity to the analyzingnano-probe. Except for this additional step using the catalyst, theprocedure to analyze the double stranded DNA is carried out using theembodiments taught herein for analyzing the single strand DNA.

While preferred embodiments have been shown and described, variousmodifications and substitutions may be made thereto without departingfrom the spirit and scope of the invention. Accordingly, it is to beunderstood that the present invention has been described by way ofillustrations and not limitation.

1-12. (canceled)
 13. A probe for analyzing an extended object, theextended object having plural sub-objects, the probe comprising a bodyhaving an edge, the edge having a thickness less than a relevantdimension of one of said sub-objects, and a length substantially greaterthan a relevant dimension of one of said sub-objects.
 14. A probe as inclaim 13 wherein said probe includes a material that hybridizes with atleast one known sub-object of said plural sub-objects.
 15. A probe foranalyzing an object, the probe comprising a body having an analyzingregion, the analyzing region having a dimension less than a relevantdimension of one (or more) of said objects, and a width substantiallygreater than a relevant dimension of one of said objects.
 16. A probefor analyzing an extended object having a plurality of sub-objects, theprobe selected from group consisting of nozzle filled with liquid, anparticle beam, electron beam, x-ray beam, a light beam, or a metal, theprobe including an analyzing region, the analyzing region having adimension less than a relevant dimension of one (or more) of saidsub-objects, and a width or a path width substantially greater than arelevant dimension of one of said objects.
 17. A probe for analyzing anobject comprising a source of a probe beam, the probe beam having ananalyzing dimension less than a relevant dimension of one (or more) ofsaid objects, and a width or a path width substantially greater than arelevant dimension of one of said objects
 18. A probe comprising a bodyportion and an active portion, the active portion having a probingdimension being a function of the thickness of a layer. 19-33.(canceled)